Seminar on antisense technology and antisense oligonucleotides converted

SEMINAR ON ANTISENSE TECHNOLOGY
AND
ANTISENSE OLIGONUCLEOTIDES
1
Department of Pharmacology BVVS COP BGK

ANTISENSE TECHNOLOGY
➢ Antisense technology is a tool that is used for the inhibition of gene
expression.
➢ The technique in which translation of mRNA into proteins is inhibited by
introducing single stranded nucleotide (Oligodeoxy nucleotides).
➢ Normal cell activity: DNA makes RNA and RNA makes protein.
➢ The no. of human disorders such as cancer, inflammatory condition and both
viral and parasitic infections result from the overproduction of normal Protein.
➢ Antisense technologies are a suite of technique that, together form a very
powerful weapon for Studying gene function and for discovering more
specific treatments of disease.
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➢ Antisense technology interrupts the translation phase of the protein production
process by,
✓ Preventing the mRNA instructions from reaching the ribosome.
✓ Inhibiting the protein synthesis.
➢ Antisense drugs are short, chemically modifies complementary nucleotide
chains that hybridize to a specific complementary area of mRNA.
➢ Antisense RNAs can be introduced directly into cells or cells can be
transfected with vectors that have been Engineered to express antisense RNA.
➢ In normal process of transcription, the double stranded DNA separates into
two strands, the sense DNA strand (Coding strand) and antisense DNA strand
(template strand).
➢ The antisense DNA strand then serves as the template for the mRNA
responsible for the code for protein synthesis in the ribosome.
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➢ The sense DNA strand may infrequently also code for RNA and this
molecules is called antisense RNA.
➢ Antisense sequences were first described as a naturally occurring event in
which an endogenous antisense RNA formed complementary to a cellular
mRNA resulting in a repressor of gene expression (Murry and Crockett,
1992).
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RATIONALE FOR ANTISENSE TECHNOLOGY
➢ The discovery that nature can regulate gene expression, and thus protein synthesis, using
antisense RNA suggested that exogenous antisense oligonucleotides might also be useful in
regulating gene expression.
➢ Antisense oligonucleotides interactions occur when the bases of the synthetic, specifically
designed antisense oligonucleotide sequence align in a precise, sequence- specific manner
with a complementary series of bases in the target mRNA.
➢ Antisense oligonucleotide interruption of the flow of genetic information may occur at the
mRNA level in the cytoplasm or by interacting with the mRNA precursor in the nucleus.
➢ Antisense RNA would be oligo-ribonucleotides that are complementary to the mRNA
sequence that is targeted.
➢ Antisense DNA would be single stranded oligo-deoxyribonucleotides that are again
complementary to mRNA.
➢ There are several mechanisms by which antisense molecules ultimately disrupt gene
expression and thus protein synthesis.
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➢ A transient inhibition may result from cross linking the oligonucleotide to the target
mRNA.
➢ The most important mechanism is appears to be through the action of an enzyme found in
most cells, ribonuclease H, which recognizes the DNA-RNA duplex (antisense RNA
interacting with mRNA), dIsrupts the base pairing and digests the RNA-part of the double
helix.
➢ Inhibition of gene expression occurs since the digested mRNA is no longer competent for
translation and resultant protein synthesis.
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❖ THERAPEUTIC ANTISENSE MOLECULES
➢ Most traditional drug molecules illicit their effect by interacting with an important enzyme or protein receptor,
antisense technology involves the blocking of genetic messages to stop the production of disease producing
proteins at the source.
➢ Antisense oligonucleotide genetic code blocking drugs might control disease by inhibiting deleterious or
malfunctioning genes, differing from gene therapy which inserts needed genetic information.
➢ Formivisen (Vitravene), the first antisense biotech drug to be approved and marketed.
➢ This nucleic acid agent is indicated for the treatment of human cytomegalovirus (CMV) induced retinitis in
AIDS pateints.
➢ Fomivirsen inhibits CMV a herpes virus, by both a base sequence specific antisense mechanism and a
sequence non-specific binding to viral coat proteins preventing CMV adsorption to host cell.
➢ As earlier, seven oligonucleotides drugs are being studied in Phase l - Phase III clinical trials.
➢ Some additional potential therapeutic targets under examination in the current clinical trials are cancer
including chronic myelogenous leukemia (CML) in accelerated phase or blast crisis, HIV infection and AIDS
and inflammatory diseases.
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❖ TRIPLEX TECHNOLOGY
➢ The term “antigen nucleic acids” has been applied to any oligonucleotides that
bind to single stranded or double stranded DNA.
➢ An antigen nucleic acid approach related to antisense technology is triple helix
(triplex) technology in which short oligo-deoxyribonucleotides of 15-27
nucleotides in length can bind sequence specifically to complementary
segments of duplex DNA.
➢ The resulting triple helices inhibit DNA replication, thus blocking genetic
information flow at the information processing level.
➢ While antisense RNA drugs would have to inhibit thousands of copies of the
synthesized target mRNA present in a cell, triplex inhibition of transcription
requires the inactivation of only one cell.
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❖ ANTISENSE OLIGONUCLEOTIDES
❖ BIOCHEMISTRY OF OLIGONUCLEOTIDES
➢ Oligonucleotides are short polymeric segments of deoxyribonucleic acid (DNA) or
Ribonucleic acid (RNA).
➢ The genes consist of double helical strands of DNA.
➢ The exact nucleic acid base sequence of the nuclear DNA contains the genetic code to make
a specific protein.
➢ Each base is linked through a phosphate bond at the 5’-position of a 2’-deoxyribose sugar
to the 3’-end of the next nucleotide.
➢ The combination of a Pyrimidine or Purine base plus a sugar moiety is a nucleoside, while a
nucleotide is a base plus a sugar moiety and a Phosphate.
➢ The specific hydrogen bonding interactions of complementary bases on each
oligonucleotide strand( A only with T and G only with C)hold the two strands of DNA
together to form double helical DNA with the sugar-Phosphate backbones directed towards
outside. Oligonucleotides are also called Oligodeoxyribonucleotides and ODNs.
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➢ The nomenclature for oligonucleotides follows a consistent pattern. For
monomer, dimer, trimer up to decamer the names would be mononucleotide,
dinucleotide etc.
➢ The name of an oligonucleotide is given by its length as a number followed by
–’mer’.
➢ Thus a 21-base containing oligonucleotide would be a 21-mer.
10

❖PHYSIOCHEMICAL PROPERTIES OF OLIGONUCLEOTIDES
➢ Normal oligo-deoxyribonucleotides and oligo-ribonucleotides containing the
five bases, unmodified sugars and the phosphate group are limited in their
potential therapeutic applications because they are highly susceptible to rapid
degradation by structurally nonspecific intracellular nucleases.
➢ Upon hydrolysis, the resulting smaller oligos and nucleotide pieces are not
expected to retain their previous biological activity nor specificity.
➢ Natural oligonucleotides have been shown to accumulate in cells by receptor-
mediated endocytosis. This process is not very efficient.
➢ Microinjection and liposome encapsulation appear to be the most effective
routes of administration of normal and modified oligonucleotides.
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❖CHEMICAL MODIFICATIONS TO ENHANCE DRUG PROPERTIES
➢ Chemical manipulation of the oligonucleotides by substituting more nuclease resistant and
lipophilic groups for the negatively charged oxygen on the phosphodiester linkages results
in a series of modified oligonucleotides with improved physiochemical properties
potentially more useful to enhance bioavailability and stability.
➢ Chemical changes of a parent oligonucleotide structure resulting in Phosphorothioate, alkyl
phosphonate and phospoamidate analogues, each possessing an additional asymmetric
center on the Phosphorus atom.
➢ These chemical changes increase lipophilicity and decrease nuclease hydrolysis.
ex: The parent oligonucleotide half-life of 1 hr is increased to more than 24 hours by
preparing the phosphorothioate derivative.
➢ A peptide nucleic acid (PNA)is a more recent variation of the phosphate sugar backbone of
the oligonucleotides.
➢ A PNA is a DNA/RNA mimic with a psuedopeptide backbone holding the pyrimidine and
purine bases in their proper spatial arrangement.
➢ Generally, aminoethyl glycine units serve as the backbone of the polymer. 12

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SYNTHESIS OF OLIGONUCLEOTIDES
➢ The synthesis of oligonucleotides can be done both in solution phase and solid phase.
➢ Solution phase synthesis refers to the synthesis in solution form while in solid phase
synthesis, one of the end of the growing nucleotide is anchored or immobilized to solid
support.
➢ Several advantages offered by solid phase synthesis over solution phase synthesis:
✓ Packing the support into a column and passing the reagents through the column allows
mechanization and automation of the synthesis process.
✓ All the reactions can be conducted in one reaction vessel. The appropriately blocked
mononucleotides are added sequentially, and the reagents from one reaction step can be
readily washed away before the reagents for the next step are added.
✓ The reagents can be used in excess in an attempt to drive the reaction completion. After
each reaction, the excess reagents can be removed simply by washing, thereby eliminating
the need for purification steps (e.g,Chromatography) between base additions.
14

✓ The different solubilities of the components can be neglected because the reaction
take place in a heterogeneous phase.
✓ Several oligonucleotides can be synthesized simultaneously in the same vessel.
✓ Individual reactions can be repeated as often as desired in order to increase yields.
➢ As compared to enzymatic synthesis, the methods for chemical synthesis of
oligonucleotides suffer from some drawbacks such as lesser efficiency than
enzymatic synthesis, size restricted to <150 bases, costlier synthesis and
purification.
➢ Different strategies for oligonucleotides synthesis:
a) Phosphoramidite method:
➢ The phosphoramidite method of DNA Synthesis is currently considered as the
standard synthesis method used in most automated synthesizers today.
➢ This method allows achieving the high coupling efficiencies needed to synthesize
longer and longer oligonucleotides with low amounts of failure sequences.
➢ The oligonucleotide phosphoramidite synthesis chemistry was introduced nearly 20
years ago (McBride and Caruthers, 1983).
15

➢ Building blocks used for synthesis are commonly referred to as “monomers”, which are
activated DNA nucleosides (phosphoramidites).
➢ The di-methoxytrityl (DMT) group is used to protect the 5’-end of the nucleoside, a
β-cyano ethyl group protects the 3’-phosphite moiety, and may also include additional
groups that serve to protect reactive primary amines in the heterocyclic nuclei bases.
➢ The protecting groups are selected to prevent branching or other undesirable side reactions
from occurring during synthesis.
➢ Oligonucleotides are synthesized on solid supports.
➢ Typically, the support is a small column filled with control pore glass (CPG), polystyrene
or a membrane.
➢ The oligonucleotide is usually synthesized from the 3’ to the 5’. The synthesis begins with
the addition of a reaction column loaded with the initial support-bound protected
nucleotide into the column holder of the synthesizer.
➢ The first nucleotide building block or monomer is usually anchored to a long chain alkyl
amine-controlled pore glass (LCAA-CPG).
16

❖A schematic diagram general outline the solid phase
oligonucleotide synthesis of a dinucleotide
(phosphoramidite method)
17

➢ The phosphoramidite approach to oligonucleotide synthesis proceeds in four
(4) steps,
➢ Automated synthesis is done on solid support, usually controlled pore glass
(CPG) or polystyrene. Synthesis is initiated with cleavage of the 5’-trityl
group by brief treatment with dichloroacetic acid (DCA) dissolved in
dichloromethane (DCM).
➢ Next, the monomer activated with tetrazole is coupled to the available 5’-
hydroxyl resulting in a phosphite linkage.
➢ Subsequent phosphite oxidation by treatment with iodine using a
THF/pyridine /H2O solution yields a phosphate backbone.
➢ The capping step with acetic anhydride, which terminates undesired failure
sequences, completes the cycle of oligonucleotide synthesis.
18

❖ Illustrates a typical synthesis cycle includes a condensation, a
capping, an oxidation, and a cleavage or deprotection step. In
general, automated DNA oligonucleotide synthesis produces a
single-stranded oligonucleotide product per column.
19

➢ Removal from the support and final base deportation process
➢ After the final sequence has been assembled, the oligomer must be removed by
cleaving it from the support and fully deprotected prior to its use.
➢ A 90 minute treatment with ammonium hydroxide at room temperature can be
used to cleave the oligomer from the support and to deprotect the phosphorous
by β–elimination of the cyanoethyl group.
➢ The acetyl capping groups and the base protecting groups are more difficult to
remove and a 24 hour treatment at room temperature or an overnight treatment at
55 °C with ammonium hydroxide allows for effective removal of these groups.
➢ After cleavage and deprotection, the resulting crude mixture contains the product
oligomer, possible truncated failure sequences with free 5’hydroxyl ends,
byproducts of deprotection, and silicates from hydrolysis of the glass support.
➢ Different purification methods can be used to separate the product
oligonucleotide from the contaminating species.
➢ H-Phosphonate method .
➢Phosphotriester method.
20

❖ APPLICATIONS OF OLIGONUCLEOTIDES
➢ Partial or total gene synthesis.
➢ Primers synthesis for DNA and RNA sequencing.
➢ Synthesis for hybridization probes for the screening of cDNA or genetic
libraries.
➢ Synthesis of adaptors and linkers for restriction site modification during gene
cloning.
➢ Oligonucleotide synthesis for application in site-directed mutagenesis.
21

MECHANISM OF ACTION OF ANTISENSE OLIGONUCLEOTIDES
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➢ In this technique, short segments of single stranded RNA are introduced.
➢ These oligonucleotides are complementary to the mRNA, which physically bind to the
mRNA.
➢ So, they block the expression of particular gene.
➢ In case of viruses, antisense oligonucleotides inhibit viral replication with blocking
expression of integrated proviral genes.
➢ These are:
✓ By base specific hybridization: which prevents access by translation machinery i.e.,
Hybridization arrest
✓ By forming RNA/DNA duplex which is recognized by nuclease RNaseH, specific or
digesting RNA in an RNA/DNA duplex.
➢ RNaseH is a non specific endonuclease, catalyzed the cleavage of RNA via hydrolytic
mechanism.
➢ RNaseH has ribonuclease activity cleaves the 3’-O-P bond of RNA in a DNA/RNA duplex.
23

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❖ LIMITATIONS
➢ Large doses are required for therapeutic response.
➢ The difficulty in directing to a particular cells.
➢ The half life in plasma is short.
❖ APPLICATIONS
➢ Antisense Oligonucleotide therapy
✓ Oncology
✓ CVS and CNS therapeutics
✓ As antiviral and antibacterial agent eg. Fomivirsen
✓ Inflammation therapeutics
➢ Other disease states like:
✓ Diabetes
✓ Amyotrophic lateral sclerosis (ALS)
✓ Asthma
✓ Hair loss
✓ Hypercholesterolemia eg. Mipomersen
✓ Arthritis
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❖REFERENCES
➢ Genetic Engineering by Smita Rastogi and Neelam Pathak.
➢ Pharmaceutical Biotechnology by Daan J A Crommelin and Robert D
Sindelar.
➢ Concepts of Genetics by William S. Klug and Michael R. Cummings.
➢ Antisense Oligonucleotide Technology in drug discovery (DOI:
10.1517/17460441.1.4.285).
➢ Antisense Drug Technology Principles strategies and Applications by Stanley.
T. Crooke.
27

THANK’S
28

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