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THERAPEUTIC ANTISENSE AGENTS AND
APTAMERS
By
NARENDAR.D
M.PHARM.
II - SEMESTER
Department of Pharmaceutics
University College of Pharmaceutical
Sciences,
KAKATIYA UNIVERSITY
Warangal - 506009
CONTENT
 INTRODUCTION
 ANTISENSE AGENTS
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




DEFINITION
MECHANISM
ADVANTAGES
CELLULAR ACTIVITY
CELLULAR UPTAKE
CLINICAL TRIAL SUBSTANCES
 APTAMERS




DEVELOPMENT
PROPERTIES
ADVANTAGES AND DISADVANTAGES
APPLICATIONS
 CONCLUSION
 REFERENCES
INTRODUCTION
The term ‘Antisense Therapeutics’ or
‘Antisense Technology’ encompasses several
types of nucleic acids that have the ability to
modulate gene expression. The most common
types of nucleic acids included in this term are
antisense oligonucleotides (ODNs), ribozymes
(RNA enzymes) and more recently, DNAzymes
(DNA enzymes).
Definition:
Antisense refers to the use of short,
Single stranded synthetic ONs to inhibit gene
expression.
 These compounds are designed to be
complementary to the coding (sense) sequence
of RNA inside the cell.
 After hybridization totarget sequences,
translational arrest occurs viaone of several
putative mechanisms.
OLIGONUCLEOTIDE
 Also called oligos.
 Sequence of DNA or RNA with a phosphate
backbone but may have a sulfate, peptide, or
morpholino backbone in place of phosphate
one, to reduce or eliminate oligo degradation
nucleases.
Main backbone of ONs is the phospodiaste.
MECHANISM
 First is the ribosomal blockade where the antisense molecule
hybridizes to the sense sequence and prevents the ribosome
from reading the mRNA code, resulting in production of a
defective nonfunctional protein.
 The second is the specific cleavage of RNA strand by activated
RNAaseH following RNA-ON hybridization. This cleavage
results in destruction of the coding message and inhibition of
protein synthesis.
 The third is the competition between the ribosome and the
antisense ON for binding to the 5’untranslated region (.5’UTR) of the mRNA.
 Binding of the ON to the 5’-UTR can also result in
activation of RNase H and subsequent cleavage of
the mRNA. Finally, synthesis of fully mature
mRNA in the cytosol can also be prevented at the
level of RNA transcription, splicing, processing. Or
transport across the nuclear membrane.
 For example, ON can bind to the complementary
sequence on nuclear DNA. forming triplex DNA
which selectively inhibits DNA transcription.
ADVANTAGES:
• Mature technology (20 years in development).
• Drug discovery and research is faster and more
predictable.
• Compounds are potentially more selective, effective
and less toxic.
• Broad disease application.
• Dosing advantages (route and frequency).
• Specificity and is the relative simplicity in which the
drugs can be rationally designed.
Antisense activity at barriers
 First, the ONs must find their way to target cells
where they must then penetrate the plasma
membrane to reach their target site in the cytoplasm
or nucleus.
 Second, once inside the cell the ON must be
able to withstand enzymatic degradation
presented by various endogenous nucleases.
 Third, the ON must be able to find and then bind
specifically to its intended target site in order to
inhibit expression of the disease-causing gene.
Stability and chemical modification
• The initial successful demonstrations of the antisense strategy
in cell culture employed the naturally occurring
phosphodiester ONs.
• Phosphodiester ONs are easily degraded in cell culture
medium containing serum due to 3’-exonuclease digestion.
Consequently, the antisense effects could only be observed if
high ON concentrations
(up to 100 PM) were used.
• Protection from degradation can be achieved by the use of a
“3’-end cap” strategy in which nuclease-resistant linkages are
substituted for phosphodiester linkages at the 3’ end of the
ON . Alternatively, ONs containing a 3’-terminal hairpin-like
structure were found to exhibit improved resistance to
exonuclease digestion.
• Phosphodiester ONs enter cells, they can be further degraded
by cellular endonucleases. Neither 3’-end caps nor 5’-end caps
protect ONs from degradation in HeLa cell extracts.
• Thus, phosphodiester ONs are poor candidates for use as
therapeutic agents in vivo. Consequently a number of
chemical modifications have been made to improve enzymatic
stability of these compounds while preserving their ability to
hybridize to cognate targets.
• The most commonly used are the first-generation analogs that
possess modifications of the phosphodiester backbone.
Examples of these include the phosphorothioate and
phosphorodithioate analogs which have sulfur
substituted for one or both nonbridging oxygens.
Cellular uptake of aptamers
 Cellular uptake of ONs is an energy-dependent process and
can be inhibited by treating the cells with metabolic inhibitors
or by lowering the temperature.
 This transport across the membrane takes place in a saturable
and sequence-independent manner. Any sequence or size of
ribo- and deoxyribonucleotide was demonstrated to compete
with labeled ON for uptake. The uptake is endocytic and
appears to be mediated by membrane receptor proteins.
 Several approaches have been developed to improve cellular
uptake of ONs. These include inclusion of ONs into liposomes
or attaching them covalently or electrostatically to specific or
nonspecific carriers.
Liposome-mediated antisense delivery
Cationic liposomes which can form stable
complexes with the polyanionic ONs. These
liposomes consist mainly of a positively charged
lipid, most notably N-[1-(2,3-dioleyloxy) propyl]N,N,N-trimethylammonium chloride or DOTMA, and
a co-lipid.
E.g. dioleylphosphatidylethanolamine, to aid
cytoplasmic delivery of the polynucleotides.
Recently, several different types of cationic lipids
have been developed including lipofectin,
quaternary ammonium compounds, cationic
derivatives of cholesterol- diacyl glycerol and lipid
derivative of polyamines
Carrier system
Mechanism
Liposomes
Cationic
pH-sensitive
Adsorptive endocytosis
Non-specific endocytosis/ Endosomal
membrane fusion
Immunoliposomes
Receptor-mediated endocytosis
Sendai virus-derived liposomes
Plasma membrane fusion
`Poly(L-lysine)
Adsorptive endocytosis
Avidin
Acridine
Adsorptive endocytosis
Intercalation
Polylysine- mediated antisense delivery
 Poly( r_-lysine (PLL), a well-known polycationic drug carrier,
has been used to facilitate cellular uptake of various drugs
including antisense ONs. Using VSV-infected L929 cells as a
model system, Lemaitre and Leonetti et al. demonstrated that
ONs complementary
to viral nucleocapsid initiation site or to viral genomic RNA
sequences promoted a sequence-specific and dosedependent antiviral activity when administered as PLL
conjugates.
 Antiviral activities of such conjugates were observed
at concentrations below 1 PM while nonconjugated ONs were
equally active when used at concentrations greater than 50
,uM. Likewise, PLL-conjugated ONs complementary to a HIV-1
splice site inhibited cytopathic effects at much lower
concentrations than non-conjugated phosphodiester or
methylphosphonate ONs.
Other methods of antisense delivery
 ON modifications reported to increase cellular uptake include the
attachment of hydrophobic molecules, such as cholesterol and
phospholipids, to the ONs.
 Coupling of a single cholesterol moiety to an ON appears to
increase its intracellular uptake by up to 1%fold.
 Similarly, anti-HIV cholesteryl-conjugated ONs are more effective
than their unmodified counterparts.
 Similarly, conjugation of an ON to phospholipids was shown to
promote its anti-tumor activity. It is not known from these studies
whether endocytosis is involved in the uptake of modified ONs.
 Avidin, a cationic protein known to internalized via an adsorptive
endocytosis process, has been coupled to ONs Association of a
biotin-conjugated ON with avidin is rapid and of high affinity.
Cellular uptake of an avidin-biotin- ON complex was shown to be
4-fold more efficient than the biotin-ON conjugate alone.
Example clinical trials studies involving antisense oligonucleotides
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
ODN
Target
Therapeutic Class
Clinical Trial hase
Company
----------------------------------------------------------------------------------------------------------------------------------------------------------Vitravene
CMV Retinitis
Anti-viral in
Approved
Isis
AIDS patients
CIBAVision
ISIS 2302
ICAM-1
Renal transplant rejection
Phase II
Isis
Psoriasis (topical)
Phase IIa
Ulcerative colitis (enema)
Phase IIa
ISIS 3521
PKC-alpha
Cancer
Phase II
Isis
ISIS 5132
c-raf kinase
Cancer
Phase II
Isis
ISIS 2503
Ha-ras
Cancer
Phase II
Isis
ISIS 14803
HCV
Hepatitis C
Phase I / II
Isis /Elan
GEM231
Protein kinase A 1a
MG98
DNA methyltransferas Cancer
Cancer
Phase II
Phase 1
Hybr idon/methylgene
Hybridon / methylgene
APTAMERS
Aptamers are artificial nucleic acid ligands that
can be generated against amino acids, drugs, proteins
and other molecules. They are isolated from complex
libraries of synthetic nucleic acid by an iterative process
of adsorption, recovery and reamplification. They have
potential applications in analytical devices, including
biosensors, and as therapeutic agents.
Also defined as aptamers are oligonucleotide
sequence that bind ligands or antigens in a way that is
similar in may respects to antibody-ligand interactions.
Aptamers range in size from approximately 6 to 40
k Da and sometimes have complex threedimensional structures, produced by a
combination of Watson–Crick and non-canonical
intramolecular interactions.
More specifically, aptamers can be classified as:
 DNA or RNA aptamers. They consist of (usually
short) strands of oligonucleotides.
 Peptide aptamers. They consist of a short variable
peptide domain, attached at both ends to a
protein scaffold.
DEVELOPMENT OF APTAMERS
 Isolation of nucleic acids from artificial libraries on the basis of
their biochemical properties were being widely discussed during
1988 and 1989, three groups independently published their results
in 1990.
 First, the Joyce group reported the use of in vitro mutation,
selection and amplification to isolate RNAs that were able to cleave
DNA.
 Second, the Gold group described experiments designed to
identify the sequence requirements of T4 DNA polymerase.
 ‘SELEX’ (selective expansion of ligands by exponential enrichment),
was able to identify the natural target of the enzyme as the
predominant, high-affinity ligand, with one major variant emerging
with similar affinity.
PROPERTIES OF APTAMERS




Structures
Aptamer Size
Aptamer Targets
Affinity
Structure:
 Determined by - enzymatic probing
- chemical probing
- NMR
- X-ray crystallography
 NMR has disadvantages
- small size and rigidity when complexed with
target.
- similariries between the interaction sites
between protien ligands and their receptrors.
Aptamer size:
• Size of aptamer depends on sequence family.
• Minimum within VEGF aptamers was between
23 and 35 nt ; minimum xanthine and guanine
aptamers were 32 nt long.
• Solvent-exposed surface area for a typical
aptamer expected to be in the range 50-60
nm2.
Aptamer targets:
• Aptamers targeted against small ions, such as
zinc, to nucleotides such as ATP,
oligopeptides and large glycoproteins such as
CD4, size range 65kDa-150kDa, with no
theoretical upper limit.
Aptamer affinity:
• Aptamers against small molecules have affinities in
micromolar range.
against aminoacids such as citrulline and arginine
range from 0.3 to 65µM, and those against ATP and
xanthine are 6 and 3.3µM respectively.
• Aptamers to nucleic acid-binding molecules have
affinities in the nanomolar range.
against retroviral integrase – 10-800nM
reverse transcriptase – 0.3-20nM
ADVANTAGES
 Used To analyze the natural processes of nucleic acid–protein
recognition.
 To generate inhibitors of enzymes, hormones and toxins with
potentially pharmacological uses.
 To Detect the presence of target molecules in complex
mixtures and to generate lead compounds for medicinal
chemistry.
 Their advantages over alternative approaches include the
relatively simple techniques and apparatus required for their
isolation, the number of alternative molecules that can be
screened (routinely of the order of 1015) and their chemical
simplicity.
Disadvantages of aptamers include:
• their pleiomorphism,
• their high molecular mass,
• the restricted range of target sites that appear
to be suitable.
IN VIVO APPLICATION OF APTAMERS
• Aptamers targeting coagulation factors
E.g against factor IXa
• Aptamers targeting growth factors or hormones
E.g against VEGF
• Aptamers targeting antibodies involved in autoimmune
diseases
E.g. auto-antibodies against nicotinic AChRs
(for
m gravis)
• Aptamers targeting inflammation markers
E.g against elastase
• Aptamers targeting neuropathological targets
E.g against synthetic βA amyloid peptide (Alzheimer)
• Aptamers against infectious diseases
E.g against gp120 or HA
• Aptamers targeting membrane biomarkers
E.g against CTL-4
• Aptamers targeting whole organisms
E.g against CMV
Conclusion
Conclusion
REFERENCES
 Y. Rojanasakul - Advanced Drug Delivery Reviews 18
(1996) 115-131
 S. Akhtar et al - Advanced Drug Delivery Reviews 44
(2000) 3 –21
 R.J. Boado - Advanced Drug Delivery Reviews 15 (1Wf)
7% 107
 James swarbrick – Encyclopedia of pharmaceutical
technology 3- edition, volume-2 935-936
 James swarbrick – Encyclopedia of pharmaceutical
technology 3- edition, volume-3 1575-1576
 William James; Encyclopedia of Analytical Chemistry; pp. 4848–4871
 S.P.Vyas and Roop K. Khar; targeted & Controlled drug
delivery: Novel Carrier Systems,
 www.pharmainfo.net.com
 www.wikipedia.org
 www.informaworld.com
Conclusion