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`MEDICINE
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`

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`

`Drug News Pcrspect 19(6), July/August 2006
`
`
`{Available on the web at: www.prouscorn/journals
`
`
` I
`
`LOOKING AHEAD
`
`The field of siRNA-based theraeutics is rapidly progressing
`with the development of chemically modified siRNAs and a variety
`of novel delivery strategies.
`
`RNA Interference
`
`for the Treatment of Cancer
`
`
`
`
`
`Summary
`RNA interference (RNAi) is the latest new technology in the field of genetic medicine
`in which specific genes can be turned off, or silenced, so as to affect a therapeutic
`outcome. it can be highly specific, works in the nanomolar range and is far more effec-
`tive than the antisense approaches popular 10—15 years ago. Here we review the field
`and explore the potential role of RNAi in cancer therapy, highlighting recent progress
`and examining the hurdles that must be overcome before this promising technology is
`ready for Clinical use. © 2006 Prous Science. All rights reserved
`___—_—_—_—_——————-——
`
`by Lisa N. Putral,
`Wenyi Gu and
`Nigel AJ. McMillan
`
`
`NA interference (RNAi) has
`received intense interest over
`
`the last few years as a potential
`therapy for infectious diseases. genet-
`ic disorders and cancer. The concept
`of using nucleic acid-based drugs is
`not new, with the potential of anti-
`sense technology reaching its heights
`in the 1990s. The idea that homolo-
`
`gous RNA sequences could be intro—
`duced into cells
`that
`inhibit
`the
`
`expression of a particular gene was
`pursued with great enthusiasm While
`antisense was effective in viii-o and
`
`its
`in validating many targets,
`useful
`therapeutic potential was never real-
`ized due to a number of
`factors,
`
`including low in vivo activity and the
`inability to deliver
`large antisense
`molecules into cells. The newest hope
`centers upon RNAi, which is more
`effective than antisense for a number
`
`including its high speci-
`of reasons,
`ficity, smaller molecule size and the
`fact
`that
`it uses a normal cellular
`
`process to ensure target mRNA degra—
`dation. While RNAi comes in a num—
`
`ber of different forms, including short-
`
`interfering RNA (siRNA), micro—
`RNA (miRNA)
`and short—hairpin
`RNA (shRNA), all achieve the same
`overall effect of silencing a specific
`
`gene. Many studies have shown RNAi
`to be effective against viruses and
`cancer cells in vitro, with the concept
`
`of using siRNA as a therapeutic drug
`becoming increasingly possible in the
`wake of research on the silencing of
`
`cancer—causing genes in vivo. A num-
`ber of delivery options for the in rivo
`delivery of siRNA to tumor tissues
`are being pursued, including topical,
`local and systemic delivery systems,
`which are discussed in detail below.
`
`However, a number of major issues
`
`must be addressed before RNAi
`
`is
`
`including its
`ready for clinical use,
`specificity, stability.
`resistance and
`delivery. Indeed, there is a feeling of
`(/e'jz‘i vu for many researchers. as these
`are the same issues that plagued the
`antisense RNA field. If these could at
`
`least be partially addressed, the future
`of RNAi—based therapy is potentially a
`bright one.
`
`A brief introduction
`
`to the mechanism of RNAi
`RNA interference (RNAi) is a form
`
`of postlranscriptional gene silencing
`(PTGS)
`in which double—stranded
`RNA (dsRNA), in tandem with protein
`complexes, catalyzes the degradation
`of complementary mRNA targets.
`It
`was first discovered in plants,‘ 3 where
`
`317
`
`Copyright © 2006 Prous Science. CCC; ()2l4—0934/2000 DOI: 10.1358/d
`@2006. l 9.0985937
`This mate-rial was copied
`at the N Lint a rid may be
`5U Eject US {bgyright Laws
`
`3
`
`

`

`LOOKING AHEAD
`
`DrLIg News Perspcct l9t6). July/August 2006
`
`
`The shRNA/siRNA
`The miRNA Pathway /’_l\t
`Pathway
`/% l
`
`
`
`bility and ultimately cell death.‘I
`However, this major stumbling block
`was overcome in 2001 when Elbashir
`
`and colleagues discovered that direct
`use of the siRNAs was successful in
`
`mammalian cells.8 The majority of
`published papers suggest that siRNA
`is a highly potent and targeted way to
`silence genes. It has proven to be an
`excellent tool for analyzing gene func—
`tion and is much more efficient than
`
`previous methods
`zymes or antisense.12
`
`involving ribo-
`
`RNAi comes in a number of dif—
`ferent
`guises
`including
`siRNA,
`miRNA and shRNA (Fig.
`l), Ulti-
`mately, Dicer processes all of them
`into siRNAs of Zlbp RNA duplexes
`with 3' 2m overhangs.I3 These siRNAs
`may be directly applied to the Zlbp
`duplex itself or by cloning a gene con—
`struct
`into a vector
`that expresses
`shRNA. shRNAs look like siRNA but
`have an added stem-loop structure,
`which is subsequently cleaved by
`Dicer to form siRNAs. shRNAs are
`
`typically expressed by plasmid vectors
`with RNA polymerase II or III pro—
`moters. An
`alternative
`strategy
`involves the delivery of siRNA via a
`viral vector such as modified retro-
`viruses and adenoviruses. The deliv-
`
`ery of plasmids expressing short-hair—
`pin RNAs is an exciting option as they
`allow “permanent” silencing of the
`target gene,
`representing a form of
`gene therapy, thereby eliminating the
`need for
`repeated treatments. Both
`shRNA and siRNAs typically have full
`complementarity to their targets and
`result
`in degradation of the target
`rnRNA, although miRNAs work by
`inhibiting mRNA translation, which
`occurs when the target
`is not corn—
`pletely complementary. miRNAs are
`normally expressed from the host
`genome,”1 although the exact mecha-
`nism of this translational inhibition in
`
`not fully understood.
`
`In vilro, the most common method
`
`for the delivery of siRNAs and shRNA
`vectors is by transfection using corn-
`rnercially available cationic lipids or
`by electroporation, neither of which
`are suitable for use in vivo. A number
`
`of options have been explored in viva,
`
`LN. Putral ct al. pp. 317—324
`
`
`
`
`
`
`Processing by l
`Drosha
`
`Pre—miRNA
`
`
`("l _ Cleavage by Dicer
`//
`'«
`
`Unwinding and
`incorporation into RISC
`
`
`
`
`
`Translational
`repression
`of target
`
`Cleavage and
`degradation
`of target
`
`A
`
`Fig.
`1. The siRNA/shRNA and miRNA pathways. These pathways share many of the same
`Dicer and RISC. miRNAs have imperfect homology to their target,
`components including
`pression, whereas siRNAs have a perfect match and lead to tar-
`resulting in translational re
`n. Following nuclear export, shRNAs and miRNAs are cleaved
`get cleavage and degradatio
`lecules with 2m 3’ overhangs. Chemically synthesized 19—21
`by DICEFi into 19-21 bp mo
`y incorporated into RISC as they mimic DICER products.
`bp siRNAs are also effectivel
`
`it was thought to protect against viral
`attack or the presence of mobile genet—
`ic elements such as transposons, both
`of which produce double—stranded or
`aberrant RNAs.’1 We now know RNAi
`is present not only in plants, but also
`in worms,5 flies‘“7 and vertebrates“)
`Moreover,
`the process is remarkably
`similar among them; briefly, dsRNA
`present in the cell becomes associated
`with the nuclease Dicer, cleaving it at
`21—23 nucleotide intervals. The result-
`ing cleavage products form a pool of
`short RNAs, referred to as short-inter-
`fering RNA (siRNA). siRNAs associ—
`ate with the multicornponent nuclease
`complex RISC (RNA-induced silenc—
`ing complex), which initiates the
`
`318
`
`unwinding of the dsRNA whereby
`either the sense or antisensc strand
`
`may enter the complex. Once loaded
`with an RNA strand,
`the RISC
`
`becomes competent to bind homolo-
`gous cellular mRNAs, which are sub—
`sequently
`cleaved
`and
`degraded
`(Fig. 1).’0
`
`Long dsRNA molecules have been
`used in plants and lower organisms
`to program the RNAi, although this
`proved ineffectual in mammalian cells
`since long dsRNAs activated the
`innate immune system and caused
`stimulation of the antiviral interferon
`
`pathway. This resulted in a generalized
`loss of protein translation, RNA sta-
`
`4
`Th is material was copiers!
`at the HLM and may be
`in bjett US Copyright Laws
`
`4
`
`

`

`it
`
`lid
`
`3.
`
`Drug News Perspect 19(6), July/August 2006
`
`LOOKING AHEAD
`
`TABLE I EXAMPLES OF RNAi AGAINST CANCER-PROMOTING GENES
`___—__'_—._—____—__—_———————
`REF.
`PATHWAYS
`TARGET GENE
`CANCER
`
`MAP kinase
`K-Ras
`Lung, ovarian, colon and pancreatic
`17—19
`H-ras
`Bladder, prostate
`20
`Nras
`Leukemia
`21
`Nox1
`Colon, prostate
`22
`b-Raf
`Melanoma
`23-26
`Skp2
`Melanoma
`25
`E6/E7
`Cervical, skin, head and neck
`27—32
`Bcr—Abl
`CML
`33
`STATS
`Breast, laryngeal
`84
`cSrc
`Colon
`35, 36
`PKC
`Breast, prostate
`20
`Bax
`37
`Lymphoma, leukemia
`Bel-2
`20, 38
`Lung
`EGFR
`39, 40
`Receptors
`Breast
`VEGF
`41
`Growth factors
`Small cell lung carcinoma, breast
`c-mye
`4243
`Transcription factors
`n—myc
`_____—________________—_—_—————
`Small cell lung carcinoma, neuroblastoma
`
`Viral oncogenes
`Mutations
`Signaling
`
`Apoptosis
`
`Abbreviations: MAP kinase, mitogen-activated protein kinase; EGFR, epidermal growth factor receptor, VEGF, vascular endothehal growth
`factor; PKC, protein kinase C.
`
`focusing on
`the earliest attempts
`hydrodynamic injection, which i11-
`volves the intravenous administration
`
`of a large volume of liquid.15 This
`technique, although somewhat effec—
`tive in rodents,
`is not translatable in
`humans. More advanced delivery sys-
`tems have followed, some of the more
`successful based upon complexing
`siRNAs into nanocarriers,
`including
`complexes composed of lipids or poly-
`mers,
`thereby protecting delicate
`siRNA molecules
`from nuclease
`
`digestion. Delivery will be discussed
`in more detail below.
`
`Potential role for siRNA
`
`in anticancer therapy
`The multifactorial nature of cancer
`
`development 21nd progression poses a
`challenge 101 the development of
`RNAi— based therapies. Cancers can
`arise from activating mutations in
`oncogenes (e.g ., Ras, e-myc),
`inacti—
`vating mutations in, or deletions of,
`tumor suppressor genes (e.g., Rb,
`p53), amplification of growth factors
`receptors (e.g., epidermal growth fac-
`tor receptor), deletion of antiapoptotic
`mediators (e.g., Bel-2), etc. Com—
`pounding this issue is the fact
`that
`individuals with the same cancer type
`can have differing genetic lesions and
`causes. In fact, there are few cancers
`for which we know the exact cause.
`Cervical cancer, of which 99.9% of
`
`cases are caused by infection with
`
`LN. l’utral et al. pp. 3l7~324
`
`human papillomavirus (HPV),16 is one
`of the rare examples.
`
`growth inhibition and apoptosis in
`cancer cells.45
`
`Despite this challenge. the specific
`targeting of particular oncogenes, or 21
`combination of multiple targets, has
`proven to be successful in a number of
`different cancers,
`resulting in the
`reduced growth or death of cancer
`cells. The list of cancer-causing genes
`targeted by RNAi
`is expanding at a
`phenomenal rate and we have chosen
`to highlight specific instances from
`various target classes (Table l).
`
`Targeting endogenous genes
`Above all, cancer cells must stay
`alive, and one means by which they
`achieve this is by overexpressing those
`genes inhibiting the cell death machin-
`ery that would normally induce apop—
`tosis. Therefore,
`these genes make
`promising RNAi
`targets A recent
`example1s that of BC-2_, a novel anti—
`apoptotic gene cha1acteiizcd by an
`siRNA-based loss-of—function screen—
`
`ing system. When BC—2 was silenced,
`apoptosis was induced in HeLa cells as
`a result of caspase-signaling pathway
`activation.“l4 Another well-cl‘taracter—
`ized feature ofcancer cells is the main—
`
`tenance of telomeres by telomerase,
`which allows the continuous replica-
`tion and evasion of senescence and
`
`apoptosis. Li and colleagues demon-
`strated that an shRNA against human
`telomerase induced p53-i11depe11de11t
`
`The targeting of specific transcrip—
`tion factors that are constitutively
`active or overexpressed in cancer cells
`is also an effective strategy. The phos—
`phoinositide 3—kinase (PI3-K) path—
`way is active in breast cancers, leading
`to repression of the forkhead box class
`0 (FoxO) transcription factors respon—
`sible for mediating growth arrest and
`apoptosis. Transfection of breast c2111-
`cer cells with siRNA against Pl3- K
`1esulted111 apoptosis and 211cductio111n
`cellular viability i11espective of
`whethe1 they we1c positive 101 the
`expression of estrogen receptor 02 or
`ErbBZ. 4“ A futthcr example is that of
`signal
`transduction and activation of
`t1ansc1iption—~3 (Stat3) by vectoidri—
`ven shRNA expression. siRNA against
`Stat3 was shown to suppress the
`growth of human prostate cancer cells,
`both in vii/'0 and in vim, via repression
`of the antiapoptotic protein Bel-2 and
`growth sti1‘1‘1ulatory proteins cyclin D
`and c-myc.47
`
`i11—
`The deregulation of genes
`volved in cell cycle control may also
`contribute to neoplastic transforma-
`tion. The role of cyclin Bl in progres-
`sion through the GZ/M phase of the
`cell cycle is well characterized, and its
`expression is known to be aberrant in
`cancer cells lines, including HeLa, as
`
`5
`This material was copied
`at the NLM and may be
`Subject U52 Copyright. Laws
`
`319
`
`5
`
`

`

`LOOKING AHEAD
`
`Drug News Perspcct 19(6). July/August 2006
`
`in a number of malignant
`well as
`[t was shown that nude mice
`tumors.
`inoculated with HeLa cells expressing
`cyclin Bl shRNA developed tumors
`with reduced growth capacity, com—
`pared with those established with
`HeLa control cells:18
`
`For tumors to become prolific and
`inetastasize, they must be well vascu-
`larized. The process of tumor angio—
`gcnesis has been investigated as an
`important
`target
`for siRNA against
`cancer. siRNA against
`the receptor
`for vascular endothelial growth fac-
`tor
`(VEGF—RZ) selectively targeted
`tumors in viva, resulting in reduced
`angiogenesis and slowed growth.”
`
`Targeting novel genes
`Ideal choices for siRNA—based
`
`therapies are cancers arising from the
`expression of a novel cancer-causing
`gene; i.e., either those stemming from
`chromosome rearrangements or from
`the introduction of exogenous genes.
`One such target is the bcr/abl fusion
`gene, expressed as a result of the
`Philadelphia chromosomal
`transloca-
`tion, and which leads to chronic
`myeloid leukemia (CML). Treatment
`of cells with bcr/abl siRNA induced
`
`apoptotic cell death in CML cells,
`whereas
`cells
`lacking this
`gene
`rearrangement were
`unaffected.33
`Other
`ideal
`targets are viral genes
`essential
`for
`the development and
`maintenance of cancer, such as the E6
`and E7 oncogenes of HPV, which
`causes cervical cancer. E6 and E7 are
`
`excellent targets. as they are essential
`for the continued proliferation of the
`cell. Moreover,
`the potential for off-
`target effects is minimized as they are
`foreign genes. A number of groups,
`including our own, have demonstrated
`that
`the silencing of E6 and E7 in
`cervical cancer cells results in a loss
`of cellular viability, resulting in either
`apoptosis or senescencePIZ‘l-‘l50
`
`Targeting mutated genes
`The targeting of mutated mRNAs
`is a viable strateg
`for silencing
`mutant genes having a single codon
`change that does not target the wild-
`type gene. This requires careful design
`
`320
`
`
`
`
`
`Targeting molecules
`
`
`
`
`Lipid bilayer
`
` &
`
`
`Liposome
`
`Nanoparticle
`
`Fig. 2. In vivo delivery systems for siRNA include liposomes and nanoparticles. Liposomes
`and nanopartieles assist in protecting nucleic from degradation in vivo. Their circulation time
`can be extended through the addition of polyethylene glycol (PEG), and they may be tis—
`sue-specific due to the addition of targeting molecules such as antibodies or peptides.
`
`and validation of the siRNA. For
`
`the oncogenic mutation of
`example,
`K-ras at valine 112 (K—ras V1”) leads
`to its constitutive activation, resulting
`in the development of pancreatic and
`colon cancers. The stable expression
`of
`siRNA against K-ras V”2 in
`CAPAN—l cells resulted in the specif—
`ic silencing and prevention of tumor
`growth in immunocompromised mice,
`yet did not effect wildtype K—ras.l7
`
`Combination therapy
`it is hoped that RNAi can be used
`in conjunction with such common
`treatment strategies as chemotherapy
`and radiation therapy. This would
`allow for the use of lower doses of
`
`highly toxic drugs or radiation, and yet
`still
`improve the overall
`therapeutic
`effect.
`Indeed,
`it has already been
`demonstrated in i»iti'()3"‘5"5-‘ and in
`
`vii/()54 that siRNAs can work synergisti—
`cally with a number of chemotherapeu—
`tic agents, enhancing the final effect.
`Furthermore, a combined treatment
`
`with radiation and siRNA targeting the
`PI3 kinase Pl 100. and p856 isoforms, or
`Akt— 1 , in cancer cell lines has also been
`
`shown to enhance cell killing.55
`
`Delivery to tumor sites
`in vivo
`
`The potential of therapeutic nucle-
`ic acid-based cancer "drugs” has been
`
`6
`Th is material was {opiad
`at the NLM anti may be
`in Diet: Ufifiopyright Laws
`
`limited by ineffective delivery sys—
`tems. The challenge of siRNA delivery
`lies
`in their
`inherent
`instability in
`serum, combined with the fact
`that
`most must be specificity targeted to
`sites of disease. Recently, numerous
`publications have described the deliv-
`cry of siRNA molecules to tumors in
`viva. Most of these are based on the
`
`use of tumor-targeted nanocarrier sys—
`tems,
`including lipoplexes and poly—
`plexes. Many such particles not only
`target
`ligands to aid tissue—specific
`uptake and polyethylene glycol (PEG)
`lipids to aid in immune cell evasion,
`but also feature extended circulation
`times (Fig. 2). Progress in this field
`has been rapid.
`
`A recent development in the use of
`siRNA as a drug has been the improve—
`ment of siRNA stability in serum.
`Chemical modifications of siRNAs
`
`have been reported to dramatically
`increase their half—life, a property that
`will increase the likelihood ofsiRNAs
`
`reaching target organs intact and func-
`tional. One example is that of locked
`nucleic acid technology (LNA), a con—
`formationally “locked” nucleotide
`analogue. siRNAs containing a num—
`ber of LNA bases exhibit a substantial
`increase in serum stability, without
`adversely
`affecting
`interactions
`with cellular silencing machinery.56
`
`l,.N. Putral et al. pp. 317—324
`
`6
`
`

`

`ix
`
`1‘l
`
`3‘
`
`LOOKING AHEAD
`Drug News Perspect 19(6), July/August 2006
`
`
`Another well—described modification
`
`is the inclusion of 2’-O-methyl RNA
`bases. It has been reported that 2’—O-
`methyl containing siRNAs maintain
`complete RNAi potency with greatly
`increased serum stability.57 The use of
`2’-O-fluoro substitutions has also been
`shown to be well
`tole‘atcd and to
`
`increase persistence in vilm.SS For all
`chemical modifications,
`the number
`and placement of modified bases is of
`critical
`importance for
`the mainte—
`nance of effective silencing.” How—
`ever, despite improvements in siRNA
`stability, naked siRNAs will be of lit-
`tle therapeutic use and are likely to
`require some form of targeted delivery
`vehicle for sufficient tissue specificity
`and cellular uptake.
`
`Delivery using cationic
`Iiposomes
`The potential use of cationic lipo—
`somes as a delivery vehicle of nucleic
`acid molecules (e.g., antisensc) has
`been explored and appears to be a
`viable option for
`the delivery of
`siRNA. Cationic lipids have been
`extensively used for the in vii/"0 deliv-
`ery of nucleic acids, although they
`have proven problematic for in vim
`i.v. administ ‘ation for a number ofrez -
`
`sons. These include rapid clearance
`from the bloodstream following injec—
`tion, distribution to primarily lung and
`liver,“ and significant toxicity due to
`activation of the innate immune sys-
`tem.“ Despite these limitations,
`the
`positive charge on cationic Iiposomes
`promotes
`uptake
`by
`angiogenic
`endothelial cells,"2 suggesting a poten—
`tial role of cationic Iiposomes for i.v.
`delivery to liver and lung tumors or for
`local administration."3 The efficacy of
`cationic liposome—delivered siRNA
`was recently demonstrated by Nogawa
`and colleagues, who targeted polo-like
`kinase—l
`(PLK-l)
`in an orthotopic
`murine model of bladder
`‘ancer,
`resulting in the inhibition of tumor
`growth.“ PLK—l
`is a highly important
`regulator of mitotic progression. and
`the elevated expression of this gene
`has been correlated to highly invasive
`bladder cancers with a poor clinical
`outcome.
`
`LN. I’utral et al. pp. 317—324l
`
`Although local delivery is appro—
`priate in some forms of cancer. a sys—
`temic approach is likely to be the only
`viable method in instances of metasta-
`
`tic cancer. Towards this goal, much
`effort is being invested in developing
`a vehicle for siRNA delivery that
`specifi ‘ally targets tumor cells that is
`nontoxic, and that may be delivered
`intravenously. For example, Chien and
`colleagues developed a
`synthetic,
`cationic cardiolipin analogtte—lmsed
`liposome (CCLA. NeoPhcctin-AT'I'M)
`to circumvent the problems associated
`with cationic lipids. This formulation
`was composed of CCLA and the fuso—
`genie DOPE lipid, entrapping siRNA
`against
`c—raf. This liposome/siRNA
`complex was able to significantly
`inhibit
`the growth of human breast
`cancer xenograft tumors in SCID mice
`by 73% following intravenous admin-
`istration,“5 They also utilized their
`CCLA to systemically deliver siRNA
`against c—raf—l to subcutaneous tumors
`derived from [’03 cells. This lipo-
`some formulation was also shown
`
`effective in inhibiting tumor growth
`and acted synergistically with the drug
`docetaxel.“ In addition, Pirollo and
`
`colleagues developed a tumor-target-
`ing immunoliposome complex con—
`sisting of the cationic lipids DOTAP
`and DOPE. linked to the antitransfer—
`
`single—chain antibody
`rin receptor
`fragment. This study did not employ
`siRNA directed against a specific
`gene, but simply characterized the
`tumor-targeting ability ofthe immuno—
`liposome/siRNA complex. This com-
`plex was shown to target both primary
`and metastatic lung tumors.“7
`
`Delivery using neutral
`Iiposomes
`Liposomes of neutral charge are
`more likely to exhibit extended circu—
`lation times and reduced toxicity in
`viva. Long-circulating Iiposomes that
`‘an avoid phagocytosis accumulate in
`tumors due to the physiological prop
`erties of tumor vasculature. As a result
`
`of tumor pathology, these vessels are
`hyperpermeable, or “leaky.” allowing
`Iiposomes to selectively extravasate to
`these tumors."8 In an example of sys-
`temically delivered siRNA demon—
`
`7
`This mate-rial was copied
`at the NLM and may be
`SL2 Eject US {beyright Laws
`
`strating functional efficacy in a mouse
`model of cancer, Landcn and col-
`
`leagues developed siRNA against
`EphA’Z, a tyrosine kinase receptor in
`the cphrin family associated with
`overexpression in human cancers with
`a poor clinical outcome. In this study,
`the neutral
`lipid DOI’C was used to
`encapsulate siRNA and liposomes
`were delivered intravenously. Lipo-
`somes were found to be 30-fold more
`efficient than naked siRNA at reduc—
`
`ing tumor weights.
`
`Delivery using polymeric
`nanoparticles
`An alternative option to Iiposomes
`is the complexation of nucleic acids,
`either siRNAs or shRNA plasmids,
`with polymeric nanoparticles.
`I’oly—
`meric nanoparticles consist of nucleic
`acids
`complexed with polymers,
`examples of which are polyethyl-
`eneimine (PEI) and poly-L-lysine.
`Targeting of nanoparticles and lipo-
`somes to specific tissues is possible
`through the addition of targeting moi—
`etics, including antibodies or their Fab
`fragments,“" peptides such as arginine-
`glycine-aspartic acid (RGD) involved
`in ligaml—integrin interactions,70 sug-
`ars. folatcs,
`transferrin and others.“
`Schiffelers and colleagues constructed
`PEGylaIed nanoparticles composed of
`PEI with an RGD peptide ligand to tar-
`get
`tumor vasculature using siRNA
`against VEGF R2, and demonstrated
`that they were effective in inhibiting
`tumor angiogenesis and growth.”
`
`Considerations
`
`for the therapeutic use
`of siRNA
`
`Although the use of siRNA for the
`treatment of cancers has great poten—
`tial, we must approach it with caution.
`The propensity of tumors to develop
`resistance to drugs is well known.
`siRNAs are highly specific, which
`means that mutations of the mRNA
`
`target in key regions may potentially
`render them ineffective. For example.
`it has been established that a single
`
`in a drastic
`base change may result
`reduction in siRNA potency.72 a prop—
`erty that a cancer cell
`is
`likely to
`exploit. For this reason, any target
`
`371
`
`7
`
`

`

`
`
`Va
`
`LOOKING AIIICAI)
`
`Drug News Perspect 19(6). July/August 2006
`
`TABLE II. siRNA-BASED DRUGS CURRENTLY IN CLINICAL TRIALS
`_——__’_——_—————
`STATUS
`TECHNOLOGY
`COMPANY
`DISEASE
`
`Phasel
`Clinical
`Phasel
`Clinical
`Phasel
`Clinical in 2006
`Second half 2006
`Phase I
`Clinical in 2006
`Phase I
`Clinical
`Phase l
`Systemic/aerosol siRNA
`Asthma
`Sirna Therapeutics
`Clinical in 2006
`___—_—_______________—______—_————
`
`Acuity Pharmaceuticals
`
`Age-related macular degeneration
`
`Alnylam Pharmaceuticals
`
`Age-related macular degeneration
`
`Alnylam Pharmaceuticals
`
`Respiratory syncytial virus
`
`Benitec
`Benitee
`
`Hepatitis C
`Human immunodeficiency virus
`
`Sirna Therapeutics
`
`Age‘related macular degeneration
`
`Modified siRNA
`
`Direct-siRNA
`
`Direct-siRNA
`
`DNA-direct siRNA
`
`Multiple siRNA. antisense
`and decoy RNA
`Modified SiRNA
`
`sequence must be carefully examined.
`and multiple targets should be consid—
`ered. Indeed, multi “warhead” systems
`have begun to appear.73 “Off target”
`effects are likely to be an issue, as it is
`known that as few as seven base pairs
`of complen'ientarity at
`the 5’ end is
`enough to confer t‘niRNA-regulated
`gene silencing.“ There is also quite 2
`high tolerance for mismatch between
`the siRNA and mRNA sequences at
`various positions within the target
`without significantly reducing silenc—
`ing capacity.75 Immune system activa—
`tion may also arise from nucleic acid
`sequences with hypomethylated CpG
`motifs, a phenomenon that may be
`enhanced when nucleic acids are com-
`plexed within Iipoplexes,70 Other
`innnunostimulatory motifs
`include
`poly (U)— and GU-rich sequences.77 It
`has been demonstrated that careful
`selection of siRNA sequences may
`reduce or eliminate innate immune
`system activation and subsequent side
`effects.77
`
`Clinical trials
`Although no clinical trials involv-
`ing siRNA against cancer are current—
`ly underway.
`at number of siRNA—
`based drugs are in phase I clinical
`trials for the treatment of various dis-
`eases including age—related macular
`degeneration. asthma and diseases of
`viral origin (Table 11).
`
`Conclusions
`Enormous strides are being made
`towards the development of siRNA—
`
`based therapeutics against cancer. The
`field is rapidly progressing with the
`development of chemically modified
`siRNAs and a variety of novel deliv—
`ery strategies. The greatest obstacle
`still to be overcome remains delivery.
`
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