aptamer as therapeutic

WELCOME

Aptamers as TherapeuticsAptamers as Therapeutics

Surender KumarP1688

(apto: “to fit”& mer: “smallest unit of repeating structure”)

single stranded folded oligonucleotides and peptide that bind to molecular (protein) targets with high affinity and specificity

Provide opportunities for structure-based drug design strategies relevant to therapeutic intervention

Can be prepared using SELEX

Aptamers combine many of the advantages of oligonucleotides and antibodies- “Chemical Antibodies”

Based on their three-dimensional structures, Aptamers can well-fittingly bind to a wide variety of targets from single molecules to complex target mixtures or whole organisms

SELEX Systematic Evolution 0f Ligands by Exponential Enrichment This method, described primarily in 1990

(Ellington, A.D & Szostak, J.W., 1990)

One of the crucial steps of a SELEX process with outstanding importance for the selection of aptamers with high affinity and specificity is the efficient partitioning between target-binding and non-binding oligonucleotides

Inorganic components Zn2+ Ciesiolka et al.

(1995)

Ni2+ Hofmann et al. (1997)

Small organic molecules Ethanolamine Mann et al. (2005)

Theophylline Jenison et al. (1994)

Malachite green Grate and Wilson (2001)

Amino acids L-Arginine Geiger et al. (1996)

L-Citrulline Famulok (1994)

L-Valine Majerfeld and Yarus (1994)

Carbohydrates Cellobiose Yang et al. (1998)

Chitin Fukusaki et al. (2000)

Sephadex Srisawat et al. (2001)

Antibiotics Kanamycin A Lato et al. (1995)

Kanamycin B Kwon et al. (2001)

Streptomycin Wallace and Schroeder, (1998)

Current opinion in chemical biology

Random DNA Oligonucleotide Library Starting point of a SELEX

consists of a multitude of ssDNA fragments (1015 molecules)

comprising a central random region of 20–80 nt flanked by different specific sequences of 18–21 nt, which function as primer binding sites in the PCR

longer random sequence pool may provide better opportunities for the identification of aptamers (Marshall and Ellington, 2000)

Selection

quantification of the enriched target-binding oligonucleotides as well as the amount of non-binding oligonucleotides of each selection round have to be determined.

Radioactive markers

(Beinoraviciute-Kellner et al., 2005; Ellington and Szostak, 1990; Shi et al., 2002)

Fluorescence labels may be used for quantification (Stoltenburg et al., 2005; Davis et al., 1997; Rhie et al., 2003)

Only few functional oligonucleotides in result of the selection step At this stage, SELEX processes for the generation of RNA and DNA

aptamers differ significantly RNA oligonucleotides

firstly have to be passed through a RT-PCR. As a result the corresponding cDNA is achieved, which is amplified in a subsequent PCR

ssDNA aptamers merely have to be amplified by PCR, where special primers can be used

to provide the aptamers with additional properties

The conditioning step is necessary to prepare the amplified oligonucleotide pool

After the preceding PCR the enriched pool is available as dsDNA. A transcription with T7 RNA polymerase has to follow in case of RNA aptamers.

The resulting RNA molecules are used as input in the following SELEX round.

ssDNA aptamers, single strand separation has to be carried out use the streptavidin/biotin. ( Fitzwater and Polisky, 1996)

the dsDNA (only one strand biotinylated) bind to streptavidin surfaces (beads or plates) and separate the strands after DNA denaturation (Naimuddin et al., 2007)

perform an asymmetric PCR which uses only one or a much bigger amount of one primer to obtain ssDNA products (Wu and Curran, 1999)

Bacterial plasmids

Sequencing dsDNA pool (thousands of sequences)

Modified plasmids

Delivery into bacteria

Only bacteria containing an aptamer insert grow.Each colony contains an individual aptamer sequence.

Bacteria Plasmids from each colony are sequenced

Sequenced Random site of aptamer sequence is unravelled

chemically modified oligonucleotide libraries

with the goal to increase the complexity of a library

To introduce new features like functional groups providing new possibilities for the

interaction with target molecules

To enhance the stability of oligonucleotide conformations

To increase the resistance to nucleases

(Jayasena, 1999; Klussmann, 2006; Kopylov and Spiridonova, 2000; Kusser, 2000)

NUCLEIC ACID MODIFICATIONSName Description Function

2’ –Aminopyrimidine Substitution at the 2’ position of a pyrimidine with NH2 group

Increased stability

2’ -O-methylpurine Substitution at the 2’ position in the sugar moiety with a methoxy group

Cheap, easy to synthesise andenzymatic incorporation

3’ and 5’ phosphorothioate caps

Replacement of an oxygen atom with a sulphur atom in the phosphate backbone to form phosphorothioate linkages and caps

Phosphorothioate linkages are chiral and form diastereomeric aptamers thus increasing stability

2’ -Fluoropyrimidine Substitution at the 2’ position of a pyrimidine with a fluorine molecule

Increased stability

2’ -Azido NTPs Substitution at the 2’ position with N3 Effective cellular internalisation

L-RNA and L-DNA L-Ribose/deoxyribose used instead of usual D-ribose thus forming mirror image aptamers

Enzymatic degradation of L-RNA/DNA is lower because of the lack of nuclease Compatibility

Locked nucleic acids (LNA)

Bicyclic ring with a furanose ring bridged between the 2’ -O, 4’-C-methylene bridge

Very high affinity to target sequences and very high stability

Name(Company)

Composition Target Indication Current Phase

Refrence

Pegaptanib sodium/ Macugen (Pfizer/Eyetech)

2′-O-methyl purine/2′-fluoro pyrimidine with two 2′-ribo purines conjugated to 40 kDa PEG, 3′ inverted dT

Vascularendothelialgrowth factor

Age-related macular degeneration

Approved in the US and the EU

Ng et al., 2006 Chakravarthy et al., 2006

AS1411/ AGRO001(Antisoma)

G-rich DNA Nucleolin Acute myeloid leukaemia

Phase II Bates et al., 2009

REG1/RB006 plus RB007(Regado Biosciences)

2′-ribo purine/2′-fluoro pyrimidine (RB006)/40 kDa PEG plus 2′-O-methyl antidote (RB007)

Coagulationfactor IXa

Percutaneouscoronaryintervention

Phase II Cooper et al., 2008

ARC1779 (Archemix)

DNA and 2′-O-methyl with a single phosphorothioate linkage conjugated to 20 kDa PEG, 3′ inverted dT

A1 domain of vonWillebrand factor

Thromboticmicroangiopathiesand carotid arterydisease

Phase II Krieg, 2006

NU172 (ARCA biopharma)

Unmodified DNA aptamer Thrombin Cardiopulmonarybypass to maintainsteady state ofanticoagulation

Phase II Sheehan & Lan, 1998

ARC1905(Ophthotech)

2′-ribo purine/2′-fluoro pyrimidine conjugated to 40 kDa PEG, 3′ inverted dT

Complementcomponent 5

Age-related maculardegeneration

Phase I Goebl et al., 2007

NOX-E36 (NOXXON Pharma)

l-RNA with 3′-PEG CCL2 Type 2 diabetes,diabeticnephropathy

Phase I Kulkarni et al., 2009

DELIVERY OF CYTOSTATICS

A specific cytotoxic aptamer–doxorubicin (Dox) conjugate. The construct is internalized via endosome, where the acidic environment favors the cleavage of the bond between the aptamer and Dox molecule. The antibiotic diffuses through the endosome membrane, penetrates the nucleus and intercalates into genomic DNA, causing cytotoxic effects.

Bifunctional conjugatesDecreases non-specific internalization of Doxgreatly enhances its uptake by target cellsthe cytotoxic effect of Dox was preserved

Data on drug biodistribution Monitoring the therapeutic effect in real time SPION– Apt–Dox conjugates

Dual Purpose Both detection and elimination of Prostate cancer cells (PCa)

A10 RNA aptamer specific for PSMA

SPION- Apt Conjugate

Imaged with NMR and displayed high specificity for PSMA-expressing LNCaP cells but not for a PMSA-non-expressing PC-3 cell line

SPION– Apt–Dox conjugates Targeted Delivery

Gold–Silver nanorods (Au–Ag NRs) high near-infrared light absorption

Aptamer Canjugates with Au-Ag NRs offers spatial precision in targeted treatment

Huang, Y. F., Sefah, K., Bamrungsap, S., Chang, H. T. and Tan, W. 2008. Selective photothermal therapy for mixed cancer cells using aptamer-conjugated nanorods. Langmuir; 24(20): 11860–5.


Aptamer-directed NRs

Efficient photothermal Convectors Promoted selective destruction of the target cells.

Efficient photothermal Convectors Promoted selective destruction of the target cells.

aptamer–liposome conjugates cargo internalization via

Fusion of membranes Endosome-mediated delivery of liposomes to the

lysosome

Major deficits1.Stability2.Nonspecific interactions which were reported during long-period incubations

Major deficits1.Stability2.Nonspecific interactions which were reported during long-period incubations

Amphipathic unit hydrophilic oligonucleotide a hydrophobic polymer In aqueous solution, self-assembled into a polarized

three-dimensional structure

Micelle system enables delivery of targeted intracellular drugs doped

inside the nanostructure as well as of therapeutic aptamers into the cell by simple membrane fusion.

Micellization could be also used as a general strategy to promote binding of low-affinity aptamers

Chimeric DNA molecules adapted to simultaneously recognize two different target proteins

Apt for CD16α Apt for c- MET

coupling with different PEG moieties and nucleotide linkers

promoted antibody dependent cellular cytotoxicity

***the bsA17 aptamer, mediated cell lysis with a magnitude similar to cetuximab***

Attractive approach to gene silencing with the aid of aptamers (Zhou et al., 2008)

Aptamer siRNA chimera targeted at HIV1 gp120 glycoprotein

selectively internalized into HIV-infected cells

Down regulated the tat/rev gene expression

Inhibitory conjugate able to suppress viremia for up to 3 weeks after the final treatment but also to evade immune response, as no significant elevation in interferon induced genes

C. In the presence of the targets the aptamers fold into their three-dimensional structures, thus opening the nanocage filled with desired molecules

A. DNA nanorobot. A nanoscale cage encapsulating cargo molecules is locked with two aptamer ‘locks’. Each combines an aptamer towards a chosen target and a complementary oligonucleotide strand

B . The cage is made of scaffold DNA fastened with multiple DNA staples

Aptamer AntibodiesEntire selection is a chemical process carried out in vitro and can therefore target any protein

Selection requires a biological system, therefore difficult to raise antibodies to toxins (not tolerated by animal) or non-immunogenic target.

Uniform activity regardless of batch varies from batch to batch.

Investigator determines target site of protein Immune system determines target site of protein.

Wide variety of chemical modifications to molecule for diverse functions

Limited modifications of molecule

No evidence of immunogenicity. Significant immunogenicity

Resistant to temperature insult Temperature sensitive

Produced chemically in a readily scalable process Not prone to viral or bacterial contamination Non-immunogenic more efficient entry into biological compartments Able to select for and against specific targets and to select against

cell-surface targets Can usually be reversibly denatured Dyes or functional groups can be readily introduced during

synthesis

Pharmacokinetic and other systemic properties are variable and often hard to predict

Small size makes them susceptible to renal filtration have a shorter half-life Unmodified aptamers are highly susceptible to serum

degradation Aptamer technologies are currently largely covered by a

single intellectual property portfolio

Aptamers can be optimized for activity and persistence under physiological conditions

during selection or during structure–activity relationship and medicinal chemistry studies

conducted after discovery

Guo et al., 2008

Addition of conjugation partners such as polyethylene glycol or cholesterol can increase

circulating half-life

Healy et al., 2004

Chemical modifications incorporated into the sugars or internucleotide phosphodiester

linkages enhance nuclease resistance

Burmeister et al., 2006

Nuclease resistance• Aptamers composed of unmodified nucleotides have half-lives in the blood that can be as

short as 2 minutes Griffin et al., 1993• Methods to overcome nuclease Susceptibility

– Modified composition aptamers• Increase purine residues

– site-specific introduction of nuclease-resistant modifications• inverting the nucleotide at the 3′-terminus• changing of the 2′-OH groups of ribose to 2′-F or 2′-NH2 groups or 2′-O-methyl substituted

nucleotides• A 3′-end capping

– streptavidin-biotin, inverted thymidine (3′-idT, creates a 3′-3′inkage) • 5′ caps

– amine, phosphate, polyethylene glycol (PEG), cholesterol, fatty acids, proteins, etc.)

(Dougan et al., 2000; Klussmann, 2006; Marro et al., 2005)• Locked nucleic acids (LNAs)

– the sugar is made bicyclic by covalently bridging the 2′-oxygen and the 4′-carbon with a methylene group

molecular mass cutoff for the renal glomerulus is 30–50 kDa

Aptamers: 5–15 kDa Methods to avoid renal filteration

Conjugation to polymers Cholesterol conjugation PEG conjugation

May lead to reduction of activity

Pharmacokinetics of aptamers conjugated to different molecular mass pegs.Pharmacokinetic profiles of 39-mer 2′-deoxy purine, 2′-o-methyl pyrimidine composition aptamers. these aptamers were unconjugated or conjugated to either 20 kda polyethylene glycol (peg) or 40 kda peg and administered intravenously to cd-1 mice (n = 3 per time point) at 10 mg per kg. data redrawn from

Fontana, D. J., Epstein, D. E. & Wilson, C. RNA as the drug discovery tool. 1. Aptamer drug development. 5). Aptamer therapeutics. Idenshi Igaku Mook 4, 61–70 (2006).

Antibodies to oligonucleotide conjugation partners Innate immune activation

TLR3 ds RNA TLR7 and TLR8 ss RNA TLR9 unmethylated CG motifs(CpG) in DNA

Anticoagulation consequence of low-affinity interactions between the

oligonucleotide and protein components of the clotting cascade

Complement activation inter action of oligonucleotides with complement factor H

Aptamers provide opportunities for structure-based drug design strategies relevant to therapeutic intervention

Recent advances in the chemical modifications of nucleic acids suggest that one of the major barriers to use, stability, can be overcome

The high affinity and specificity of aptamers rival antibodies and make them a promising tool in diagnostic and therapeutic application

We should expect more aptamers to be isolated in the near future against an ever increasing repertoire of targets, using these different SELEX approaches with increased speed and efficiency

Aptamers are poised to successfully compete with monoclonal Abs in therapeutics and drug development within the next few decades

Keefe, A. D., Pai, S and Ellington, A. 2010. Aptamers as therapeutics. nature reviews, 9:537-50.


Stoltenburg, R., Reinemann, C. and Strehlitz, B. SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands Biomolecular Engineering 24 (2007) 381–403

Nimjee, S. M., Rusconi, C. P. and Sullenger, B. A. Aptamers: an emerging class of therapeutics. Annu Rev Med 2005, 56:555-583.

aptamer as therapeutic

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