Aptamers are small nucleic acid molecules that bind target molecules with high affinity and specificity. They are generated through an in vitro selection process called SELEX and have several advantages over antibodies such as low immunogenicity and toxicity. Recent advancements have improved SELEX technology and clinical development of aptamer-based therapeutics is ongoing for cancer, eye diseases, and other applications. However, challenges remain around nuclease degradation, renal filtration, controlling duration of action, and potential toxicity that must be addressed for aptamers to reach their full potential as targeted therapeutics.

1. APTAMERS AS TARGETED THERAPEUTICS: CURRENT
POTENTIAL & CHALLENGES
Presented By:
Dibya Sundar Padhy
19PCM2980
MS (Pharm.), Sem III
Dept. of Pharmacology & Toxicology
NATIONAL INSTITUTE OF PHARMACEUTICAL EDUCATION AND RESEARCH
S.A.S. Nagar, Punjab

2. INTRODUCTION
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 Aptamers are small (usually from 20 to 60 nucleotides) single-stranded RNA or DNA oligonucleotides
able to bind target molecules with high affinity and specificity.
 Aptamer: aptus = to fit (Latin) + meros = part (Greek)
 Aptamers are nucleotide analogues of antibody.
 RNA aptamers provide a significantly greater structural diversity compared to DNA aptamers.
 The conventional method for aptamer engineering known as SELEX.
(Systematic Evolution of Ligands by Exponential enrichment)
 Tuerk & Gold first described the SELEX process in 1990.
Bouchard et al., Annu. Rev. Pharmacol. Toxicol. 2010; 50; 237-257

3. ADVANTAGES OF APTAMERS
High affinity & specificity
Neither immunogenic nor toxic
Very small – good tissue penetration
Easily chemically modifiable for:
 Increased stability
 Reduced toxicity
 Combination therapy
Easily manufactured (<60 nt)
Easy scale-up
Rapid in vitro discovery- SELEX
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4. SELEX - SYSTEMATIC EVOLUTION OF LIGANDS BY
EXPONENTIAL ENRICHMENT
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• Purified protein
• Whole cell
• Live animal
Scheme of SELEX
Jiehua jhou et al., Nature reviews 2017; 16; 181-202

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Live animal based SELEX
Jiehua jhou et al., Nature reviews 2017; 16; 181-202

6. RECENT ADVANCEMENTS IN SELEX TECHNOLOGY
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o Efficient partitioning and recovery
o Negative selection
o Specialized partitioning technologies- CE, AFM, flow
cytometry, microfluidics, biacore SPR
o Accurate amplification
o Emulsion PCR (ePCR)
o Droplet digital PCR (ddPCR)
o Global analysis of sequencing data
o High throughput sequencing (HTS) technology
o HT-SELEX
Jiehua jhou et al., Nature reviews 2017; 16; 181-202

7. RECENT PROGRESS IN APTAMER BASED THERAPEUTICS
• Aptamer as Antagonists/Inhibitors.
o HIV 1
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Tat protein
TAR in the HIV 1 LTRs
Regulates viral
transcription
o Targeted inhibition of Prostate cancer metastasis with an RNA aptamer to prostate
specific membrane antigen (PSMA).
Jiehua jhou et al., Nature reviews 2017; 16; 181-202
Synthetic RNA decoy
Mimics

8. CONT…
• Aptamer as Agonists.
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Aptamers Nature Target Effect
4-1BB
OX40
CD28
RNA aptamers T cell
costimulatory
receptors
Cancer
immunotherapy
IR-A48 DNA aptamer Insulin receptor Stimulates
glucose uptake
CD40* RNA aptamer SMG1(a kinase)
by conjugating an
shRNA
Activation of B-
lymphocyte
*Bivalent aptamer Agonist
Monovalent aptamer Antagonist
(Reduces B cell lymphoma proliferation)

9. CONT…
• Aptamers as Delivery agents.
o Therapeutic oligonucleotides
RNAi (siRNAs, shRNAs, miRNAs)
Aptamers
Antisense
o Drug conjugates
Small molecule drugs
Proteins
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10. CLINICAL DEVELOPMENT OF APTAMER-BASED THERAPEUTICS
Drug name/ Company Target
Therapeutic
purpose
Clinical trials
(Current status)
Condition being
treated
Macugen® (Pegaptanib
sod.)
(Pfizer/ Eyetech)
VEGF165
Macular
degeneration
US FDA
Approved(2004)
AMD, Diabetic
macular edema
NOX-E36
(NOXXON Pharma)
Human
chemokine
CCL2
Type 2
diabetes
Phase III
Diabetic
nephropathy
NOX-A12
(NOXXON Pharma)
Stromal cell-
derived
factor1(SDF1)
Oncology Phase II
Multiple myeloma, non-
Hodgkin’s lymphoma,
Chronic lymphocytic
leukaemia
REG1 anticoagulation
system
(Regado Bioscience)
Coagulation
factor IXa
Coagulation Phase III
Acute coronary
syndrome, Coronary
artery disease
(CAD)
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11. LIMITATIONS & CHALLENGES
Limitations
in Aptamer
Neuclease
degradation
Renal
filtration
Toxicity
Duration of
action
control
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Lakhin, A.V. et al.; Aptamers: Problems, solutions and prospects. Acta Naturae 2013, 5, 34–43.

12. NUCLEASE DEGRADATION
The rapid degradation of aptamers (especially RNA aptamers) by nucleases in
biological media, and in blood.
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Base
Modification at 2’ position of
monosaccharide (Resistance to
endonuclease)
Modification of 3’-terminus
(resistance to 3’-exonucleases)
Modification of 5’-terminus
(resistance to 5’-exonucleases)
 Development of Spiegelmers or “Mirror aptamers”. (L-ribose or L- deoxy ribose)

13. RENAL FILTRATION
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 Most aptamers have a molecular weight ranging from 5 to 15 kDa (15–50 nucleotides).
 Susceptible to renal filtration
 As the molecular mass cutoff for the renal glomerulus is 30–50 kDa.
 Aptamers that are conjugated to polymers in this size range show significant reduction
in renal filtration rates. The most commonly used polymer is PEG.

14. CONTROL OF THE DURATION OF ACTION
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 The duration of action depends on multiple factors:
 Degradation
 Involvement in metabolic processes
 Renal excretion
 It can be solved by generating antidotes to aptamers by synthesizing a complementary
oligonucleotide.
Aptamer
(Active)
Antidote Complex
(Inactive)
 Application of polycationic biopolymers. (Porphyrin)
 Inducible activation
Lakhin, A.V. et al.; Aptamers: Problems, solutions and prospects. Acta Naturae 2013, 5, 34–43

15. TOXICITY
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 Aptamer related adverse effects are rare in clinical evaluation to date.
 Polyanionic effects –
 Unexpected tissue accumulation
 Non-specific immune activation (Continuous / Repeated administration)
 Unnatural nucleotides may cause chemical toxicity & becomes immunogenic.
Eg. 2’-fluoropyrimidine modified RNAs cause hepatotoxicity.
 Associated with the formulation of therapeutic aptamers.
Eg. Serious allergic response to PEG group (Reported in Phase III study of aptamer based anticoagulant system)
 Highly lipophilic molecules
Non-specific liver uptake
Hepatotoxicity
Jiehua jhou et al., Nature reviews 2017; 16; 181-202

16. CONCLUSION
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 Aptamers are a special class of substances that combine the advantages both of low-molecular-weight substances
and proteins.
 Aptamers demonstrate an affinity and specificity similar to those of monoclonal antibodies. Meanwhile, aptamers
are non-immunogenic and demonstrate high tissue penetration similar to that of small molecules. However,
aptamers have not been commonly used thus far.
 The aptamer generation protocol SELEX was developed appx. 30 years ago, but only one aptamer, Macugen (or
Pegaptinib), has been approved for therapeutic application.

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Immunotherapy
Cardiovascular diseases Neurological diseases
Anti-viral therapy
Eye diseases
Cancer
Diagnostics

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