Aptamers and antisense oligonucleotides

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DEPARTMENT OF PHARMACEUTICSMOLECULAR PHARMACEUTICS
PRESENTED BY:
SAGAR.G
M. PHARM, 2nd SEM
DEPARTMENT OF PHARMACEUTICS
SUBMITTED TO :
Dr.B. WILSON
HEAD OF THE DEPARTMENT
DEPARTMENT OF PHARMACEUTICS
APTAMERS AND ANTISENSE OLIGONUCLEOTIDES 
 
NUCLEIC ACID BASED THERAPEUTIC DRUG DELIVERY SYSTEMS

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•Aptamers (from the Latin aptus – fit,
and Greek meros – part) are single stranded
oligonucleotide or peptide molecules that bind to a
specific target molecule
• 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
Aptamers
•Aptamers assume a variety of shapes due to their tendency to form helices
and single-stranded loops. Hence, they are extremely versatile and bind targets
with high selectivity and specificity •Common targets include proteins,
peptides, carbohydrates, small molecules and many other compounds 
•Aptamers can be used for both basic research and clinical purposes as
macromolecular drugs 
• Aptamers can be combined with ribozymes to self-cleave in the presence of
their target molecule

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•Aptamers are usually selected from the oligonucleotide collection that
is known as the initial oligonucleotide pool (IOP) and includes 1014-1015
different oligonucleotides. IOP is often called a “combinatorial library”
•IOP is an aliquot of the synthetic chemical combinatorial library and contains
single-chained DNA or RN A oligonucleotides conditioned for binding to the target
molecule. 
•The conventional method for aptamer engineering known as SELEX (Systematic
Evolution of Ligands by Exponential Enrichment)
Aptamers can be classiﬁed as
1. DNA or RNA or XNA (Xeno nucleic acid) aptamers
They consist of (usually short) strands of oligonucleotides.
2. Peptide aptamers
They consist of one (or more) short variable peptide domains, attached at both
ends to a protein scaffold.

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Nucleic acid Aptamers
•Nucleic acid aptamers are nucleic acid species that have been engineered
through repeated rounds of in vitro selection or equivalently, SELEX
(Systematic Evolution of Ligands by Exponential Enrichment) to bind to
various molecular targets such as small molecules, proteins, nucleic acids,
and even cells, tissues and organisms
•Aptamers are useful in biotechnological and therapeutic applications as
they offer molecular recognition properties that rival that of the commonly
used biomolecule, antibodies
•In addition to their discriminate recognition, aptamers offer advantages
over antibodies as they can be engineered completely in a test tube, are
readily produced by chemical synthesis, possess desirable storage
properties, and elicit little or 
no immunogenicity in therapeutic applications

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Structure of an RNA aptamer speciﬁc for biotin. The aptamer surface
and backbone are shown in yellow. Biotin (spheres) ﬁts snugly into a
cavity of the RNA surface

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Peptide Aptamers
•They are are artificial proteins selected or engineered to bind
specific target molecules
• These proteins consist of one or more peptide loops of variable
sequence displayed by a protein scaffold
• They are typically isolated from combinatorial libraries and often
subsequently improved by directed mutation or rounds of variable
region mutagenesis and selection
• In vivo, peptide aptamers can bind cellular protein targets and
exert biological effects, including interference with the
normal protein interactions of their targeted molecules with other
proteins.
•Libraries of peptide aptamers have been used as "mutagens", in studies in
which an investigator introduces a library that expresses different peptide
aptamers into a cell population, selects for a desired phenotype, and identifies
those aptamers that cause the phenotype
• The investigator then uses those aptamers as baits, for example in yeast two-
hybrid screens to identify the cellular proteins targeted by those aptamers

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•The Affimer protein, an evolution of peptide aptamers, is a small, highly stable
protein engineered to display peptide loops which provides a high affinity binding
surface for a specific target protein.
•It is a protein of low molecular weight, 12–14 kDa, derived from the cysteine
protease inhibitor family of cystatins
•The Affimer scaffold is a stable protein based on the cystatin protein fold. It
displays two peptide loops and an N-terminal sequence that can be randomised to
bind different target proteins with high affinity and specificity similar to
antibodies
Affimer
•Stabilisation of the peptide upon the protein scaffold constrains the possible
conformations which the peptide may take, thus increasing the binding affinity
and specificity compared to libraries of free peptides.

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Aptamers can be used in:
1. Afﬁnity reagents
2. Bioimaging probes
3. Biosensing
4. Therapeutics, e.g. Pegaptanib
Applications

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Applications
Antibody replacement
• Aptamers have an innate ability to bind to any molecule they're targeted at,
including cancer cells and bacteria 
•Bound to a target, aptamers inhibit its activity. Aptamers suffer from two issues
that limit their effectiveness. Firstly, the bonds they form with target molecules ar
usually too weak to be effective,and second, they're easily digested by enzymes 
•Adding an unnatural base to a standard aptamer can increase its ability bind to
target molecules
•A second addition in the form of a "mini hairpin DNA" gives the aptamer a stable
and compact structure that is resistant to digestion, extending its life from hours
to days 
•Aptamers are less likely to provoke undesirable immune responses than
antibodies

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1. Aptamer degradation
•The rapid degradation of aptamers (especially RNA aptamers) by nucleases
in biological media, and in blood in particular, is a serious problem that puts
limits on their practical application
•The average time of oligonucleotide decay in blood ranges from several
minutes to several tens of minutes depending on the oligonucleotide
concentration and conformational structure
• Since such a short time range is unacceptable for most therapeutic
applications, several methods for protecting aptamers against degradation by
nucleases have been developed.
2. Aptamer excretion from the bloodstream by renal ﬁltration
•The removal of aptamers from the bloodstream via renal ﬁltration complicates
their therapeutic application 
•Most aptamers have a molecular weight ranging from 5 to 15 kDa (15-50
nucleotides), and they can be easily excreted by kidneys capable of removing
substances with a molecular weight below 30-50 kDa
• Conjugation of aptamers with polyethylene glycol (PEG) with a molecular
weight of 20 or 40 kDa is the most common solution to this problem
Limitations

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3. Interaction of aptamers with intracellular targets
•Most aptamers were selected using molecules located on the cell surface or in the
bloodstream 
• This potentially makes their application rather easy, since all that is needed to
trigger the therapeutic effect is to deliver the aptamer into the bloodstream
• However, some advances in the intracellular delivery of aptamers have recently
been achieved 
• Special expression systems are able to generate aptamers inside cells and
ensure their accumulation either in nucleus or in the cytoplasm
4. Control of the duration of action
•The pharmacokinetic parameters of a drug (e.g., action duration) are very
important in its therapeutic application. The duration of action depends on
multiple factors, including degradation, involvement in metabolic processes, renal
excretion, etc
• All these factors should be taken into consideration before drug prescription,
and sometimes they limit its application 
• The use of aptamers as drugs can often solve the problems associated with
controlling the duration of action
• One of the possibilities is to generate antidotes to aptamers by synthesising a
complementary oligonucleotide

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5. Aptamer cross-reactivity
•Regardless of their high specificity, aptamers that recognise
particular targets can also bind to molecules with a similar structure 
•Aptamer cross-reactivity can be an obstacle to their therapeutic
application because of the possible side effects caused by aptamer
interaction with other proteins; however, this problem can be avoided
by introducing a SELEX negative selection step with structurally
similar molecules
6. Automation of aptamer generation
•Generation of aptamers seems to be a rather simple protocol,
but in reality it is a time- and labor-consuming process
• The selected aptamers sometimes turn out not to have the best
affinity and specificity due to a suboptimal SELEX procedure
• Automated SELEX allows one to avoid these problems and to
generate aptamers with the required qualities within several days
•Another new method known as CE -SELEX (capillary
electrophoresis SELEX) includes a modified stage of selection of
target-bound oligonucleotides and allows to generate aptamers
in one round

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Current status of aptamers in diagnostics & Therapy
1. Mono- and polyclonal antibodies are routinely used for the diagnostics of various
diseases. However, they can sometimes be successfully replaced by aptamers,
especially when effective and speciﬁc binding to a target molecule is required
2. Aptamers can recognise a membrane-immobilized protein in Western blotting
protocols more effectively than antibodies ca
3. ELISA protocols are also more sensitive when aptamers are used instead of
4. Similar to antibodies, aptamers can be used to purify target proteins
5. In contrast to antibodies, aptamers can be selected against non- immunogenic
and toxic substances
6. Aptamers are also used as recognizing elements in biosensors. They are
10-100 times smaller than antibodies and can be arranged with a higher density
on the biosensor surface. Aptamer-based biosensors require a smaller volume of
the tested sample and can be re-used without loss of sensitivity
7. Aptamers are promising therapeutic agents, because they are cheap, non-
immunogenic, and easy to modify

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8. Inhibition of target enzymes is the main field of aptamer application as drugs.
Aptamers inhibit target enzymes by binding to the catalytic center or inducing
conformation changes in a protein’s structure
However, when an aptamer is similar to an activating ligand, it can induce enzyme
activation
9. Aptamer-based protocols of treatment of viral diseases are under
development. Aptamers that recognise many viruses, including the human
immunodeficiency virus (HIV), hepatitis C virus (HCV) and influenza virus,
are already available
10. Aptamers against cell-type specific protein markers can be conjugated to
drugs for targeted delivery
11. The aptamer generation protocol SELEX was developed over 20 years ago,
but only one aptamer, Macugen (or Pegaptanib), has been approved for
therapeutic application. Macugen binds to the vascular endothelial growth
factor (VEGF) and blocks abnormal angiogenesis in the eye, thus preventing
intraocular hemorrhage and loss of vision

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antisense oligonucleotide
Small pieces of DNA or RNA that can bind to speciﬁc molecules of RNA. This
blocks the ability of the RNA to make a protein or work in other ways.
Antisense oligonucleotides may be used to block the production of proteins
needed for cell growth. They are being studied in the treatment of several types
of cancer. Also called antisense agent.
• Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded oligo
deoxynucleotides that can alter RNA and reduce, restore, or modify protein
expression through several distinct mechanisms
• By targeting the source of the pathogenesis, ASO-mediated therapies have an
higher chance of success than therapies targeting downstream pathways
• Advances in the understanding of ASO pharmacology have provided momentum
for translating these therapeutics into the clinic
• Two ASO-mediated therapies have received approval from the US Food and Drug
Administration for the treatment of Duchenne muscular dystrophy and spinal
muscular atrophy
• Further advancement of ASOs in the clinic urgently requires optimization of
ASO delivery, target engagement, and safety proﬁle
• This technology holds the potential to change the therapeutic landscape for
many neurological and non-neurological conditions in the near future

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Mechanisms of action for therapeutic oligonucleotides.
(A)control of splicing by ASOs and alternative splicing. In contrast to gapmers,
ASOs that control splicing possess ribose or morpholino modifications
throughout the oligomer;
(B) block of translation by antisense gapmers (oligonucleotide that contain 2ʹ-
modified RNA flanking a central DNA region) targeting mRNA;
(C) block of translation by a fully complementary dsRNA and RNA interference.
Mechanism of action
(A) RNase H cleavage induced by (chimeric)
antisense‐oligonucleotides
(B) Translational arrest by blocking the ribosome.

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First generation Antisense oligonucleotides:
First synthesised by Eckstein and colleagues in 1960s .
Phosphoro-thioate -deoxynucleotides are the first generation oligonucleotides
and have a sulphur atom replacing the non-bridging oxygen of the sugar
phosphate backbone. It preserves the overall charge and can also activate
RNaseH for the degradation of mRNA.
Second generation Antisense oligonucleotides :
Second generation Antisense oligonucleotides containing nucleotides with alkyl
modifications at the 2’ position of the ribose.
2’-O-methyl and 2’-O- methoxy -ethyl RNA are the most important member of
this class.
These “second-generation” oligonucleotides are
resistant to degradation by cellular nucleases
and hybridise specifically to their target mRNA with higher affinity than the
phosphodiester or phosphorothioate.
However, such antisense effects result from RNase H independent mechanisms.

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Third generation Antisense oligonucleotides
Newest and most promising.
Enhance binding afﬁnity and biostability. Peptide nucleic acids (PNAs) 
Locked nucleic acid (LNA) Tricyclo-DNA (tc DNA)
Cyclohexene nucleic acids (CeNA)
Delivery of oligonucleotide
Antisense oligonucleotides penetrate into the targeted cells through active
transport
Oligonucleotides endocytosis has shown to be mediated by the nucleic acid
specific receptors
Cationic liposomes have used to protect ASO and to ease their entry into the cell
These liposomes have high affinity for negatively charged cell membranes and
are delivered by endosomal pathway into cells

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Antisense technologies
These are techniques used for studying gene function and for discovering new
specific treatments of diseases
In Antisense technology, synthetically produced molecules seek out and bind
to messenger RNA (mRNA), blocks the final step of protein production
mRNA is the nucleic acid molecule that carries genetic information from the
DNA to the other cellular machinery involved in the protein production
By binding to mRNA, the antisense drugs interrupt and inhibit the production
of specific disease-related proteins
Inserting antisense into cells
Endocytosis
One of the simplest methods to get nucleotide in the cell
It relies on the cells natural process of receptor mediated endocytosis
The drawbacks to this method are the long amount of time for any
accumulation to occur, the unreliable result, and the inefficiency
Micro-infection
The antisense molecule would be injected into the cell
The yield of this method is very high

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Liposome encapsulation
This is the most effective method, but also a very expensive one.
Liposome encapsulation can be achieved by using products such as
lipofect ACE to create a cationic phospholipids bilayer that will
surround the nucleotide sequence. The resulting liposome can merge
with the cell membrane allowing the antisense to enter the cell.
Electroporation
The conventional method of adding a nucleotide sequence to a cell
can also be used. The antisense molecule should transverse the cell
membrane offer a shock is applied to the cells.

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Application of Antisense Oligonucleotides:
Antisense drugs are being researched to treat a variety of diseases such as :
1. Lung cancer
2. Colorectal carcinoma
3. Pancreatic carcinoma
4. Malignant melanoma
5. Diabetes
6. Amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy Asthma, 
Arthritis.

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Aptamers and antisense oligonucleotides

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