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595
ISSN 1757-6180
Bioanalysis 
(2009) 1(3), 595–609
10.4155/BIO.09.66 © 2009 Future Science Ltd
R
eview
Oligonucleotide (OGN) biopharmaceuticals arecurrently being investigated at preclinical andclinical stages or various disease indications. Todate, omivirsen, an
antisense
 
oligonucleotide(ASO) phosphorothioate (PS) targeted at cyto-megalovirus retinitis, and pegaptanib, an anti-angiogenic pegylated
aptamer
or the treatmento neovascular age-related macular degeneration,have been approved by the US FDA in 1998 and2004, respectively 
[301,302]
.Investigational nucleic acid-based therapeu-tics occupy an increasingly important spacein the biopharmaceutical drug-discovery and-development landscape. It is expected thatmore OGN drugs will reach the market, con-sidering the relative potency o later generationcompounds, such as
siRNA 
 
[1]
, the capacity or controlled manuacturing o OGN andthe appealing prospect o simple and eectivedrug design.Oligonucleotide drug candidates requirerobust bioanalytical assays or their determina-tion in increasingly complex biological matrices,such as skin, colon or brain tissue. In addition,sensitive assays are required or the lower thera-peutic doses that more eective drug regimensand targeted delivery will bring about. Thebioanalytical method platorm is also careully selected based on the structure and unction o the
therapeutic OGN
. Numerous classes o therapeutic OGN compounds exist, including:
n
siRNA 
[2,3]
n
 ASO OGNs
[4]
n
 Aptamers
[5]
n
Immunomodulatory OGNs (IMOs)
[6,7]
n
miRNA-blocking OGNs
[8]
n
RNA decoys
[9]
n
Splice switching/exon skipping OGNs
[10]
n
Ribozymes and DNA enzymes
[11]
Current investigational therapeutic OGNs aretypically either siRNA, ASO, IMOs or aptamers. ASOs are complementary to a targeted, disease-associated RNA molecule, generally a mRNA.The classical ASO mechanism is based on trig-gering RNase H cleavage o the ASO–RNA tar-get duplex
[4]
. ASOs pioneered the therapeuticOGNs, and a number o OGN chemistries weredeveloped or ASO that have been adapted oruse with other OGNs.Toll-like receptors (TLRs) are pattern-recognition receptors and recognize moleculescommon to bacteria and viruses. TLR9 isexpressed in two types o cells o the immunesystem: plasmacytoid dendritic cells and B cells.IMOs containing unmethylated CpG motismimic nonmammalian DNA and thereore bindto the TLR9, thereby inducing proinammatory cytokines and Th1-type immune responses
[6,7]
.They are agonists o the innate immune system. Aptamers are OGNs that have been selected via
in vitro
molecular evolution techniques
[5]
. They are ligands or specifc molecular targets, mostly proteins o therapeutic relevance. Aptamers aretypically selected using an iterative molecu-lar evolution technique known as SystematicEvolution o Ligands by Exponential enrich-ment (SELEX)
[12]
. They are habitually designedto work at the extracellular level.Recent advances with RNAi and siRNA syn-thetic compounds have ueled interest in thera-peutic OGNs in recent years. Andrew Z Fire and
Bioanalysis of siRNA and oligonucleotide
therapeutics in biological uids and tissues
This article summarizes bioanalytical avenues for the determination of siRNA and oligonucleotide therapeutics,with an emphasis on hybridization methods. Aspects of the chemistry and delivery of investigational oligonucleotidetherapeutics are considered. The nature of the oligonucleotide under investigation will dictate the best analyticalcourse of action; each method has its advantages and disadvantages, depending upon the oligonucleotide test article
and the anticipated toxicokinetic and pharmacokinetic study parameters. Stringent method development and specicvalidation criteria are essential to attain the best quality results in support of a regulatory ling.
Guy A Tremblay
1
&
Philip R Oldfeld
1†
†
Author for correspondence
1
Immunochemistry, CharlesRiver Preclinical & ClinicalServices, 22022Transcanadienne, Senneville,QC H9X 3R3, Canada,Tel.: +1 514 630 8263Fax: +1 514 630 8230
E-mail: philip.oldeld@crl.com
A
ptAmeR
Oligonucleotide ligand obtainedby
in vitro
evolution
A
ntisense
Complement strand of atargeted nucleic acid,typically mRNA
si
RnA
19–25-nucleotide longdouble-stranded RNAmolecules involved in theRNAi pathway
t
heRApeutic
 
oligonucleotide
 
A class of oligonucleotidesconsisting of approximately
10–40 DNA, RNA or modied
nucleic acid monomers used fortherapeutic applications
 
Bioanalysis 
(2009) 1(3)
596
future science group
Craig C Mello were awarded the 2006 NobelPrize or their discovery o RNAi gene silenc-ing by double-stranded RNA 
[303]
. RNAi is anatural mechanism o RNA inhibition medi-ated by small, double-stranded RNA moleculeso 19–25 nucleotides. The RNAi feld expandedast within academia, where siRNA molecules were designed to knock-down the expression o aselected gene in cell culture. It has been reportedthat a proper selection o the target sequence may typically lead to a knockdown o approximately 75% o a given mRNA and its protein productthereo 
[1]
.Synthetic siRNAs have been demonstrated totarget genes
in vivo
or multiple diseases, includ-ing ovarian
[13]
and bone cancer
[14]
, hypercholes-terolemia
[15]
, liver cirrhosis
[16]
, respiratory syn-cytial virus
[17]
, hepatitis B virus
[18]
and humanpapillomavirus
[19]
.Delivery and modiication are importantareas o ocus or OGN drug development, sincethey can improve upon aspects o tissue-specifctargeting, cell entry, stability and potency.Improved cell-specifc targeting and transec-tion efciency will help to lower the eectivedose. For those OGNs that act at the extracel-lular level, robust modifcation chemistries thatenhance the hal-lie and solubility o the OGNin plasma will be necessary in order to preventdegradation by circulating nucleases.Quantitative, highly specifc and sensitivebioanalytical methods are required to deter-mine the toxicokinetic (TK)/
pharmacokinetic
 
(PK)
 
parameters and exposure–response inorder to choose the right dosage regimens o therapeutic OGNs in support o their preclini-cal and clinical development. The methods will be used to measure OGN concentrationsin plasma, urine, bile, eces and solid tissues,typically kidney, liver, brain and spleen; butother target tissues, such as skin and vitreoushumor, have also been investigated.
Chemistry of investigationalOGN therapeutics
Several modifcations to investigational thera-peutic OGNs have been introduced over theyears. They can involve the phosphodiesterlinkage group, the ribose sugar moiety or thenucleotide bases. Termini can be modifed as well; or example, additions to the 5´ or 3´ endso OGNs with polyethylene glycol (PEG) oraptamers
[20]
, cholesterol or siRNA 
[21]
orpeptides or exon skipping OGNs
[22]
havebeen developed.For therapeutic OGNs to be part o clini-cal trials, they are required to be robust, saeand eective.
F
iguRe
1
depicts native RNA 
(F
iguRe
1A)
and modifcation chemistries used with investigational therapeutic OGNs.The approved ASO therapeutic omiversen hasa phosphorothioate (PS) oligodeoxynucleotide(ODN) chemistry. PS ODNs are the frst-genera-tion ASOs intended to increase the nuclease resis-tance and cellular uptake o the phosphodiesterbackbone
in vivo
 
[23]
. The PS modifcation, wherea nonbridging oxygen in the phosphodiester link is substituted with sulur
(F
iguRe
1B)
, impartsconsiderable stability to PS ODNs
in vivo
. ThePS group also coners binding afnity towardsproteins
[24]
, which may help to protect the OGNrom circulating nucleases
[25]
.The pegaptanib aptamer, the other approvedtherapeutic OGN, is pegylated at the 5´ end andhas an inverted 3´-3´deoxythymidine residue atthe 3´ end. Pegaptanib is 2´-
O
-methylated (2´
O
-Me) on purines and 2´-uorine-modifed (2´F)on pyrimidines
[26]
.The 2´-F-modifed
(F
iguRe
1c)
 
nucleic acidderivatives adopt the A-orm typically ound inRNA, bind targets with high afnity and aremore resistant to nucleases
in vivo
 
[27]
. The 2´Fis a useul modifcation or aptamers, since the2´F residues can be incorporated by RNA poly-merases used with
in vitro
molecular evolutiontechniques
[28]
. O note, the RNAi pathway istolerant to 2´F derivatives, which also makesthe modifcation useul or siRNA therapeuticapplications
[29]
.The 2´
O
-Me RNAs
(F
iguRe
1d)
are second-generation ASO modifcations and coner con-siderable protection rom nucleases while havingsimilar hydrogen bonding properties to RNA–RNA hybrids
[23]
. An interesting property o 2´
O
-Me is related to their propensity to abrogate theinherent immunostimulatory characteristics o OGNs when added selectively to guanosine oruridine residues o a siRNA 
[30]
, considering theobservation that the siRNA under investigationmay unction via an unspecifc immune-stimu-latory mechanism
[31]
. Interestingly, the 5-meth-ylcytosine (5mC) nucleobase modifcation, natu-rally ound in CpG motis, has also been shownto reduce immunogenicity or a PS ODN
[32]
.Typically used in combination withPS, 2´-O-methoxy ethyl (2´MOE;
F
iguRe
1e
)-modifed ODNs are also second-generation ASO modifcations
[33]
. In additionto supporting RNase H cleavage
[34]
, OGNs withthe 2´MOE modifcation have increased afnity 
p
hARmAcokinetics
Describes the relationshipbetween mechanisms of drugabsorption, distribution andelimination over time
R
eview
|
Tremblay & Oldeld
 
www.future-science.com
597
future science group
towards target RNA and increased nuclease sta-bility relative to unmodifed phosphoramidate(PN) ODNs
[35]
.One example o a second-generation moleculemodifed with PS and 2´MOE in clinical devel-opment is OGX-011, an ASO designed to block the production o clusterin, a cell-survival pro-tein that is overproduced in several cancer indi-cations
[36]
. OGX-011 is complementary to themRNA translation initiation site and has beenshown to inhibit clusterin expression in
in vitro
 and
in vivo
laboratory models
[37]
.
Figure 1. Chemical modifcations developed or investigational therapeutic oligonucleotides.
OOOHOO
-
OOPbaseOOOO
-
SOPbase OO FOO
-
OOPbasebaseOOO OCH
3
O
-
OO
P OO OOO
-
OOPbaseOCH
3
OOObaseOO
-
OOP
A. RNAB. Phosphorothiate oligodeoxynucleotideC. 2´-fluoro RNAD. 2´-
O 
-methyl RNAE. 2´-
O 
-methoxy ethylF. Locked nucleic acidG. Morpholino oligomer
ONOOPH
3
CNOObaseH
3
C
Bioanalysis of siRNA & oligonucleotide therapeutics in biological uids and tissues
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