In the one decade that have passed since the discovery of RNA
interference (RNAi), efforts and huge money have been invested in
research to find out the therapeutic application of gene silencing in
humans and the number of publications related to RNAi have also
increased exponentially. There is encouraging data from ongoing clinical
trials for the treatment of age-related macular degeneration and
respiratory syncytial virus. A significant advantage of this siRNA-based
technology is the rapidity with which different siRNA sequences and the
matching genes can be studied, particularly useful for drug target
validation. However, the major challenge in navigating these molecules
for regulatory approval is inefficient delivery to the target site
resulting in serious off target effects and immunological responses. The
widespread use of RNAi therapeutics for disease prevention and
treatment requires the development of clinically suitable, safe and
effective drug delivery vehicles. This review highlights the potential
of siRNAs in gene silencing over other technologies, the various
challenges encountered during in-vivo siRNA delivery with possible
strategies to overcome them and a view to the prospect of siRNAs in
therapeutics.



Gene
therapy involves gene replacing, swapping, repairing or silencing of a
nonfunctional or transmuted gene to treat diseases such as cancer, HIV,
etc. Gene silencing means "switching off" of a particular gene by a
mechanism other than genetic modification and can be achieved by
distinct strategies viz. ribozyme technology, antisense
oligonucleotides, aptamers and RNAi (figure 1). Within a short span of
time, the use of RNAi has rapidly spread to nearly every aspect of
biomedical research from target validation to therapeutics. siRNAs,
shRNAs and the most recently discovered miRNAs are the functional
mediators of RNAi.

siRNAs are double stranded RNA fragments
generated by the cellular enzyme Dicer, typically comprising of 19 to 23
nucleotides with a molecular weight of approximately 13 to 15 kD and a
negative charge of 38 to 46 mV.

Dicer processes dsRNAs into 21-25
nucleotide siRNAs which are subsequently incorporated into one or more
of the Argonaute proteins in RNA-induced silencing complex (RISC) where
the RNA serves as a sequence specific guide for complementary base
pairing with the target and guides RISC for sequence specific target
degradation or translational inhibition.

For gene silencing
activity intracellular entry into the target cell within the diseased
tissue and subsequent appearance in the cytoplasm to silence the target
mRNA is required for the siRNAs. However, primary obstacles for
achieving this in vivo include competitive uptake by nontarget cells,
excretion in urine, degradation by nucleases, and endosomal trapping.
Overwhelming efforts have been made and are still in progress for the
development of promising siRNA therapeutics by chemical manipulations,
designing appropriate delivery vector, using targeting ligands, or a
combination thereof. This critique summarizes the potential of siRNAs as
therapeutics, challenges in siRNA delivery along the possible
strategies used to overcome these with a view to the future of siRNAs in
the management of chronic diseases.

Why siRNAs?
siRNA
based RNAi technology has been acknowledged by researchers for its
therapeutic potential as a potent, highly specific and cost-effective
way of treating diseases by inhibiting a target protein far before its
formation before translation. The leeway of chemical modification and
designed synthesis has opened enormous possibilities of using siRNAs as
potential therapeutics. Most drugs provide only a symptomatic relief
from a disease rather than destroying its root cause i.e. the disease
causing protein. Even cell-specific drugs like monoclonal antibodies
have limited targets and act mainly on circulating proteins or
cell-surface receptors.

Compared to other antisense strategies
siRNAs are highly specific efficiently blocking the production of the
disease causing protein before it is translated. A potential aspect of
siRNA therapeutics is that they can be precisely tailor made to access
unlimited number of intracellular targets with the knowledge of
sequenced human genome and delivery methodologies. RNAi is an innate
biological response, and hence represents a more natural strategy for
manipulating gene expression making use of siRNAs and miRNAs. Further,
certain diseases are caused by mutation in a single allele with siRNAs
targeting specifically the disease-causing mutation leaving the normal
allele intact.

Compared to other antisense agents, siRNAs have
longer half life and are effective at very small concentrations avoiding
the need of repeated administrations. Most viral diseases are
manifestations of changing viral mutations that limits the use of an
antiviral drug against a particular mutant only. A blend of mutant
specific siRNAs in a single delivery vector is a wonderful remedy for
such mutating diseases. Furthermore, considering multi drug resistance
associated with chemotherapeutic and antibiotic therapies, siRNAs can
specifically inhibit the expression of resistance causing proteins.

Thus,
siRNAs enjoy a sound position in antisense therapy being easy
amenability to target specific designing, specific and potent with the
ease of administration through various routes like pulmonary, ocular,
nasal, oral, intracerebral, intramuscular, intravenous, intratumoral,
etc. Table 1 summarises the key features of siRNAs and other gene
silencing approaches. However, the major challenge to siRNA delivery
in-vivo is inefficient delivery to the target site and can be
acknowledged by better understanding of the various barriers encountered
as per its path from the site of delivery to the site of action.

Challenges in siRNA delivery
Degradation
by nucleases Following administration, the first biological barrier
encountered by siRNAs is presented by the nuclease activity in the blood
and tissues resulting in low gene silencing. Chemical modifications at
the 2’-OH position of pentose sugars or 3’ half of the siRNA structure,
and use of suitable delivery vectors can drastically improve the
stability of siRNAs towards nucleases. The substitution of sulphur for
oxygen in phosphorothioate and 2’-OH modifications like in locked
nucleic acids (LNAs), peptide nucleic acids (PNAs), etc. improve
biological stability of siRNAs towards nucleases. Further, the inclusion
of 6-carbon sugar instead of ribose and 2’-F and 2’-OMe modifications
and use of gapmers also prolong the biological half life of siRNAs in
vivo. Furthermore, the formulation of lipoplexes and polyplexes protect
siRNAs from degradation by nucleases.

Glomerular Filtration, Hepatic Metabolism and RES Uptake
Molecules
less than 70kDa and 5nm in diameter undergo glomerular filtration and
siRNAs being small readily get excreted soon after their administration.
Also, following administration siRNAs easily get phagocytosed by the
mature macrophages residing in the tissues of reticuloendothelial system
(RES) like liver, spleen and lungs. While, particles smaller than 100
nm leak from the intercellular junction of capillary endothelium to the
hepatic interstitial spaces due to hepatic uptake and get trapped by the
Kupffer cells there.

Colloidal complexes of siRNA with cationic
polymers or lipids get destabilized as aggregates due to the presence of
serum proteins, platelets, and RBCs in the blood. Thus, both the size
and charge of these complexes determine their clearance from the body. A
coating of polyethylene glycol neutralises the surface charge and
imparts a protective hydrophilic sheath around these complexes making
them long circulating. Thus, clearance and RES uptake of siRNAs can be
avoided by carefully manipulating the size and charge of the final
complex to around 100nm and near to neutral respectively.

Endothelial barrier
The
endothelial cells that line the vascular lumen present a barrier to
siRNA delivery as need to cross the endothelium before being delivered
to the tissue parenchymal cells. siRNAs being large and negatively
charged can not readily cross the cellular membranes. However, certain
tissues such as liver and spleen own relatively larger endothelial
intercellular spaces than any other body tissue allowing access of even
large siRNA molecules. They can follow either paracellular route through
the intercellular pores, or the transcellular pathway which is
claveolin based transcytosis, to egress across the endothelial lining.
Cell penetrating peptides, targeting ligands or molecular conjugates can
be used to facilitate passage of siRNAs across the endothelial lining.

Cellular uptake and subcellular distribution
For
gene silencing activity siRNAs must undergo cellular uptake and sub
cellular distribution to degrade the target mRNA in the cytoplasm.
Transcellular pathway through receptor mediated endocytosis is the main
mechanism of cellular uptake and can be facilitated by conjugating the
siRNAs with a receptor specific ligand such as monoclonal antibodies or
cell penetrating peptides like penetration, transportation, etc. After
cellular uptake sequential intracellular trafficking into the
subcellular compartments occurs and finally, siRNAs must be released
from the endosomes to reach the target mRNA in the cytoplasm to effect
gene silencing and is usually achieved by conjugating endosomal release
signal peptides to siRNA entrapped within a nano carrier.

Immunological barrier
Our
body has inbuilt mechanism to fight against invading foreign bodies
conferred by two distinct mechanisms namely innate immunity and adaptive
immunity and acts as a barrier to in vivo siRNA delivery. The innate
immunity is of major concern in case of naked as well as viral vectors
based siRNA delivery. Also, adaptive immunity mediated by T and B
lymphocytes is of concern as interacts with specific surface receptors
on T and B lymphocytes causing activation and production of effector T
cells and antibodies against the invading foreign bodies. Researchers
have reported serious immune responses with siRNA therapeutics.

Current status
Currently,
various siRNA-based drugs are under clinical investigations (Table 2),
although expressed short hairpins and at least one anti-miRNA antisense
are in trials. The siRNAs in clinical trials are by far chemically
synthesized, bypassing the Dicer cleavage step for entry into RISC. Only
two clinical trials use ex vivo delivery, whereas most of the trials
employ systemic delivery including injections directly into the target
tissues such as the eyes for treatment of age-related macular
degeneration or directly into tumours, inhalation or infusion of siRNAs
incorporated in delivery vectors. However, there is no FDA approved
siRNA drug till date, this may change within the next few years, and
will open doors for more approved siRNA therapeutics.

Currently
siRNAs are enjoying too many clinical applications for diseases. In
clinics the safety assessment of siRNAs is more complex than classical
drugs being an endogenous mechanism. However, there are no safety
guidelines addressing siRNA till date with only a little information
available from research engines.

Future envisaged
siRNAs
have demonstrated promising results in early stages of clinical trials
against mutating diseases. Today, siRNA based formulations are developed
with patent point of view which is likely to boost investments in this
arena. siRNAs use the endogenous mechanism of RNAi and we can expect
maximal therapeutic benefit from siRNA delivery. Similarly, it can be
used in almost all kind of diseases and can lead to personalized
medicines. Many clinical trials have been initiated since last one
decade after first report on siRNA use as therapeutics.

Pharma
giants across the globe have joined hands to successfully deliver siRNAs
in vivo and looking for glorious future. However, scenario in India is
still gloomy and needs attention of Indian multinational pharmaceutical
companies to collaborate with research/academic institutions to gear up
the research in this rewarding area of research to reap the benefit of
potential of siRNA in regular clinical practice for prevention,
cure/biosensitization of available drugs in otherwise presently
incurable diseases.
We expect to see siRNAs as standalone
therapeutics in future, but also will be used in combination with
current therapies for alleviation of human sufferings. Thus, a combined
knowledge of human genome sequence, targeted delivery systems, and siRNA
designed therapies will be used in future therapies including/against
any mutating disease. Although, the systemic delivery of siRNA to
specific targets and their availability in cytoplasm of pathogenic
tissue still remains greatest challenge and needs attention of the drug
delivery scientists.

Authors are faculty, Pharmacy Det, The Maharaja Sayajirao University, Baroda,Gujarat