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CONTENTS

• INTRODUCTION
• CELL SPECIFIC APTAMERS
• SELEX
• RECENT ADVANCES IN CELL-SELEX
• DNA AND RNA AS APTAMERS
• MOLECULAR BASIS OF APTAMER BINDING
• ANTIBODIES Vs APTAMERS
• APTAMERS FOR THERAPY
• APTAMERS AVAILABLE AS FUTURE DRUGS
• LIMITATIONS OF APTAMERS
• CONCLUSION
• REFERENCES

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INTRODUCTION

• Aptamers are short, 50-100 base-long,
  oligonucleotides capable of recognizing a wide
  range of target molecules, and bearing a group of
  characteristics important for the development of
  novel diagnostic and therapeutic strategies.
• They can be composed of RNA, DNA or modified
  bases, and are selected from vast populations of
  random sequences, through a combinatorial
  approach known as systematic evolution of ligands
  by exponential enrichment(SELEX)
• Aptamers have been selected for various purposes,
  making use of their high specificity, versatility
  and affinity in target recognition.

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CELL SPECIFIC APTAMERS

• Aptamers for extracellular targets have high
  potential for diagnostic and therapeutic
  applications.
• In therapy, aptamers for extracellular targets
  can be used directly as effectors (activators or
  inhibitors) or indirectly as vehicles for drug
  delivery.
• Cell-SELEX is an evolutionary approach, and thus
  allows the selection of aptamers even without
  prior knowledge of specific targets.
• Aptamers can, therefore, speed up the discovery
  of new biomarkers that are per se targetable.

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SELEX

• SELEX is an iterative process used to identify an
  aptamer to a chosen molecular target.
• The process is constituted of three main phases
• 1.Selection phase
• It is designed to identify those molecules
  with the
• greatest affinity for the target of
  interest.
• 2.Partition phase
• It is designed to physically separate the
  aptamertarget complexes
• from the unbound molecules in the mixture,
  effectively separating
• the true binders from the weak or
  non-binders.
• 3.Amplification phase
• In this phase, the captured, purified
  sequences are enzymatically
• amplified using PCR amplification, to
  generate a new library of molecules
• that is substantially enriched for those
  aptamers that can effectively bind
• the target.

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RECENT ADVANCES IN CELL-SELEX

• In many cases, binding to a specific cell-type
  can be achieved by selecting aptamers for an
  abundantly presented protein on the cell-surface
  using traditional protein SELEX.
• Blank et al. used endothelial cells as targets
  and selected DNA aptamers that could be used as
  histological markers of microvessels in brain
  tumours.
• Most technical advances of the SELEX procedure
  focused on improving the individual steps of the
  process, in particular the actual selection step,
  in order to make the generation of aptamers as
  fast and efficient as possible.
• FACS (Fluorescence Activated Cell Sorting) was
  used to remove the fraction of dead cells and to
  select aptamers binding to live cells. Thus, they
  obtained aptamers specific for B-cells that were
  able to differentiate between different cell
  subpopulation.

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DNA AND RNA as APTAMERS

• RNA or DNA usually create a specific cavity to
  enclose and recognize a small molecule, or adapt
  to the enclosure of a bigger molecule.
• The detection of the first nucleic acid aptamers
  to a protien that does not normally interact with
  RNA or DNA was a single stranded DNA aptamer to
  thrombin, indicating the beginning of a new class
  of thrombin inhibitors.
• RNA has intrinsic properties that lead to the
  formation of a variety of elaborate structures,
  some of which are able to catalyse reactions and
  others are able to function as receptors and
  ligands.
• Single stranded DNAs are rarely encountered in
  nature, and even less frequently do these single
  stranded DNAs form shapes, other than the usual
  duplex, that serve a functional role.

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MOLECULAR BASIS OF APTAMER BINDING

• The molecular characteristics of aptamer
  interactions with a variety of molecular targets
  have been studied using nuclear magnetic
  resonance spectroscopy(NMR).
• Aptamers show interactions in the following
  manner
• 1.Aptamers are folded into unique overall
  shapes to form intricate
• binding pockets to accommaodate their
  targets.
• 2.Functional groups scattered on an aptamer
  are brought in close
• proximity to form a cluster of molecular
  forms that specify target
• interaction.
• 3.Aptamers discriminate molecules that are
  closely related to
• cognate targets at the atomic level.
• The objective of many aptamer selections for
  protein targets is to recover DNA molecules that
  specifically bind enzymes or receptor proteins
  and interfere with the receptor pathway.

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ANTIBODIES vs APTAMERS
ANTIBODIES APTAMERS
1.Antibodies are produced biologically in a process that is difficult to scale up without affecting product characteristics. 2.Viral or bacterial contamination of manufacturing process can affect product quality. 3.Often immunogenic. 4.Susceptible to irreversible denaturation limited shelf life. 1.Aptamers are produced chemically in a readily scalable process. 2.Chemical production process is not prone to viral or bacterial contamination. 3.Non-immunogenic. 4.Can usually be reversibly denatured, and phosphodiester bond is extremely chemically stable.
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APTAMERS FOR THERAPY

• Aptamers are not only a new and promising
  alterative to antibodies in diagnostics, they may
  also be used in therapy and medicine.
• Aptamers as Blocking Agents Aptamers might be of
  therapeutic relevance due to their inhibitory
  effects on disease-related cell-surface
  components.
• One promising approach would be the application
  of aptamers acting on deregulated processes in
  inflamed tissues or pathological vasculature
  during angiogenesis.

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• Aptamers as Stimulating Agents McNamara et al.
  introduced agonistic aptamers specific for the
  costimulatory receptor 4-1BB, a member of the
  tumour necrosis factor (TNF) receptor family.
• This receptor is presented on activated CD8
  T-cells and prolongs the survival and expansion
  of CD8 T lymphocytes.
• Since CD8 T-cells play an important role in
  tumour immunity, enhancing 4-1BBs costimulatory
  activity could support anti-tumour immune
  responses.

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• Aptamers for Delivery of Toxic Payloads Aptamers
  can also serve as modules that selectively
  recognize and bind to defined cell types or
  tissues.
• By appending drug molecules, the aptamers can be
  used to deliver cargo molecules to or into
  specific cells or tissues of interest.
• Prostate Specific Membrane Antigen (PSMA) One of
  the best studied aptamers binds to the prostate
  specific membrane antigen, a well-known tumour
  marker for prostate cancer.

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• Glycoprotein 120 (gp120) Envelope glycoprotein
  gp160 of HIV-1, consisting of two parts, gp120
  (glycoprotein 120) and gp41(glycoprotein 41),
  respectively, plays a crucial role in HIV-1
  infection.
• Therefore, blocking HIV-1 entry into T-cells with
  the aid of corresponding aptamers seems to be a
  therapeutically promising strategy.
• James et al. reported the selection of
  2-F-modified RNA aptamers that bound to gp120
  with high affinity, and thereby neutralized the
  infectivity of R5 strains of HIV-1 in human
  peripheral blood mononuclear cells by blocking
  the interaction.

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• Nucleolin Nucleolin, a multifunctional
  eukaryotic nucleolar phosphoprotein, plays a
  pivotal role in RNA transcription, in DNA
  replication, and in the regulation of different
  steps of ribosome biogenesis.
• Furthermore nucleolin binds to the mRNA of Bcl-2,
  an antiapoptotic protein overproduced in several
  cancer types, and thereby increasing its
  half-life.
• Nucleolin is overproduced in the cytoplasm and on
  the plasma membrane of certain tumour cells.
• Epidermal Growth Factor Receptor (EGFR) The
  epidermal growth factor receptor is a
  transmembrane receptor with intrinsic tyrosine
  kinase activity. EGFR functions in a wide range
  of cellular processess, including cell
  proliferation and apoptosis and overproduced in
  some type of cancer cells.
• Li et.al reported in 2010 on the selection and
  application of aptamers that bind specifically to
  EGFR via endocytosis.

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• Transferrin Receptor (Tfr) The enzyme
  replacement therapy (ERT) is a therapeutic
  application to replace an absent or deficient
  enzyme in patients by a functional counterpart.
• Receptors that underlie endocytosis are highly
  relevant for escort of these lysosomal enzymes
  into cells if coupled to appropriate delivery
  vehicles like aptamers. One of the mentioned
  receptors is Tfr, ubiquitously presented on
  mammalian cells.
• It directs iron to cells via binding of
  transferrin-Fe3, followed by cellular
  endocytosis and iron release into the endosomes.

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APTAMERS AVAILABLE AS FUTURE DRUGS

• 1.Pegatinib was approved for therapeutic use by
  the US Food and Drug Administration in
  December2004 and is currently marketed by Pfizer
  and Eyetech as Macugen.
• It is a VEGF-specific aptamer that binds to
  all isoforms of human VEGF except for the VEGF121
  and is administered by intravitreal injection of
  0.3mg per eye once every 6 weeks, and is used to
  ameliorate the loss of visual acuity that is
  caused by the aberrant angiogenesis that is
  characteristic of age-related macular
  degeneration(AMD).

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• 2.REG1 is an anticoagulation system that includes
  RB006, a coagulation factor IXa-specific aptamer,
  and its oligonucleotide antidote RB007.
• REG1 is currently being evaluated in the
  clinic as a reversible anticoagulant for use
  during percutaneous coronary intervention.
• It is being developed by Regado Biosciences
  and is currently in Phase II clinical trials.

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• 3.ARC1779 binds to the A1 domain of von
  Willebrand factor and inhibits the capacity of
  this domain to bind to platelet membrane
  glycoprotein Ib receptors, thereby eliciting an
  antithrombotic effect without significant
  anticoagulation.
• It is being developed by Archemix and is
  currently in Phase II clinical trials for
  thrombotic microangiopathies and in patients with
  carotid artery disease undergoing carotid
  endarterectomy.
• 4.NU172 aptamer is currently being evaluated in
  Phase II clinical trials by ARCA Biopharma.
• It is capped, substituted or conjugated to
  PEG, it has a short duration of action in vivo
  and is intended to be given by continuous
  infusion during cardiopulmonary bypass or other
  surgical procedures to maintain a state of
  anti-coagulation with a rapid return to
  haemostasis once the infusion ceases.

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• 5.AS1411 formerly AGR0001, is thought to elicit
  its therapeutic effects through its interaction
  with nucleolin.
• It is being developed by Antisoma and is
  currently in Phase II clinical trials for acute
  myeloid leukaemia, but it has recently been
  decided not to continue clinical evaluation of
  AS1411 for renal cancer.
• 6.E10030 binds to platelet-derived growth factor
  (PDGF), which is known to play a role in the
  recruitment and maturation of pericytes that can
  increase resistance to the anti-VGEF treatment of
  AMD.
• It is being developed by Ophthotech and is
  currently in Phase I clinical trials.

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LIMITATIONS OF APTAMERS

• Pharmacokinetic and other systemic properties are
  variable and often hard to predict.
• Small size makes them susceptible to renal
  filtration and they therefore 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.

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CONCLUSION

• Since the invention of the SELEX process morethan
  two decades ago, considerable progress has been
  made in the field of aptamer technology.
• Aptamers for all kinds of targets, ranging from
  small molecules to cell surface receptors have
  successfully been selected.
• Aptamers are formidable delivery tools, because
  they are comparatively small and can easily
  penetrate tissues or become cointernalized with
  the receptors they are specifically binding to.
• Aptamers can be tuned for desired applications
  modified bases can be integrated to increase the
  in vivo stability or to regulate their in vivo
  function.

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REFERENCES

• A. D. Ellington and J. W. Szostak, In vitro
  selection of RNA molecules that bind specific
  ligands, Nature, vol. 346, no.6287, 1990, pg nos
  818822.
• P. R. Bouchard, R.M.Hutabarat, and K. M.
  Thompson, Discovery and development of
  therapeutic aptamers, Annual Review of
  Pharmacology and Toxicology, vol.50, 2010, pg nos
  237257.
• S. D. Jayasena, Aptamers an emerging class of
• molecules that rival antibodies in
  diagnostics, Clinical Chemistry, vol.45, no. 9,
  1999, pg nos 16281650.
• A. D. Keefe, S. Pai, and A. Ellington, Aptamers
  as therapeutics,
• Nature Reviews Drug Discovery, vol. 9, no.
  7, 2010, pg nos 537550.

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• M. Blank, T. Weinschenk, M. Priemer, and H.
  Schluesener,Systematic evolution of a DNA
  aptamer binding to rat brain tumor microvessels
  selective targeting of endothelial regulatory
  protein pigpen,Journal of Biological
  Chemistry,vol. 276, no.19, 2001, pg nos
  1646416468.
• T. C. Chu, J. W. Marks, L. A. Lavery et al.,
  Aptamertoxin conjugates that specifically
  target prostate tumor cells, Cancer Research,
  vol.66, no. 12, 2006, pg nos 59895992.
• H. Liu, P. Moy, S. Kim et al., Monoclonal
  antibodies to the extracellular domain of
  prostate-specific membrane antigen also react
  with tumor vascular endothelium, Cancer
  Research, vol. 57, no. 17, 1997, pg nos
  36293634.

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