CREDIT SEMINAR (VPT-600)

TOPIC : NUCLEIC ACID DRUGS

DEPARTMENT OF VETERINARY
PHARMACOLOGY AND TOXICOLOGY

MAYANK GUPTA
I.D. No. 44241

OVERVIEW

Introduction
Antisense oligonucleotide (ASONs )
Aptamers
Ribozymes
DNAzymes
siRNAs
Antigene nucleic acid compounds
Cellular uptake
Delivery systems
Conclusion
Future Prospects

INTRODUCTION
The dream of modern drug research
Target specific drugs
Efficient drugs with no side effects
The problem with the conventional therapies
Binds to non-target proteins
Produces side effects

Central
dogma of
molecular
biology

 Nucleic acid drugs turn off genes by

targeting the RNA that codes for the protein

instead of the protein product itself.
RNA is an ideal target for drug discovery

because it is an important mediator of gene

expression.
By synthesizing complementary sequences

ANTISENSE
OLIGONUCLEOTIDES
Zamecnik and Stephenson (1978) were the

first to propose the

oligonucleotides
for
purposes .

Antisense

use of synthetic
the
therapeutic

describes
the
interaction
between oligonucleotides complementary
to (sense) pre-mRNA~ or mRNA molecules;
these
inhibit
the
oligonucleotides
production of the protein product.

Mechanism of
Action

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Design of ASONs
Length :
optimal length

12 to 25 bases.
Longer than 25 bases
difficulty in cellular
uptake.

Hybridization Capacity:
Antisense oligonucleotides are designed to
hybridize with the mRNA of interest.

Backbone Modifications.
To improve stability.

The most common modifications include

Introduction of

Phosphorothioate
Methyl phosphonate
hydrophobicity

stability

Modified ASONs
First generation ASONs
Second generation
ASONs
Third generation ASONs

First generation ASONs

Major representatives
Most widely used ASONs

Phosphorothioate (PS)
oligodeoxynucleotides

Advantages of Phosphorothioate
Antisense ODNs (PTOs)
- Excellent stability against exo- and
endonuclease
(half life >48hours in serum)
- Excellent solubility
- Highly specific hybridization characteristics
- Low toxicity
- Established synthesis procedure and fast
production
-Only PTOs approved as antisense drugs

Second generation ASONs

Nucleotides with alkyl

modifications at the
2' position of the
ribose.

2'-O-methyl and 2'-

O-methoxy-ethyl RNA

Less toxic than

phosphorothioate
DNAs and have a
slightly enhanced
affinity towards their
complementary RNAs

Third generation ASONs

Zwitterionic ASO
Positive charged functional groups are

added to oligonucleotides polyanionic

backbone can be partially neutralized,
resulting in less electrostatic repulsion
of the two strands and, in theory,
better membrane permeating abilities.

Third generation ASONs

Peptide nucleic acids (PNAs),
N3 P5 phosphoroamidates (NPs),
2-Deoxy-2-fluoro--d-arabino nucleic acid

(FANA)
Locked nucleic acid (LNA)

Therapeutics of ASONs
Fomivirsen:
First Antisense Drug
Cytomegaloviral induced

retinitis

Two antisense drugs in

Clinical trial (Phase III)

Genassense by Genta
; Bcl2 inhibition
ISIS 3521 : Inhibition
of Protein kinase C-
Effective against cell
lung cancer

APTAMERS
Aptamers
selected and amplified oligonucleotides
isolated from random pools of synthetic

oligonucleotides
Aptamers directly inhibit a proteins function

by folding into a specific three-dimensional

structure that dictates high-affinity binding
to the targeted protein.

SELEX
(systematic evolution of
ligands by exponential
enrichment)
process for production
of aptamers

Mechanism of Action
of
APTAMERS

The beginning
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Formation of complex with delivery system

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Interaction between transfection-capable

complexes and negatively charged cell
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membrane

Receptor mediated endocytosis

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Endosome formation
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Endosome breakdown
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Release into cytoplasm

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Delivery system dissociation

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Interaction with proteins

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Inhibition of disease
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Advantages of APTAMERS
Bind their targets with dissociation constants (Kd) in

the low picomolar to low nanomolar range.

Aptamers display low to no immunogenicity.

Entire selection is a chemical process carried out in

vitro and can therefore target any protein

Uniform activity regardless of batch synthesis

Unlimited shelf-life
Aptamer-specific antidote can be developed to

reverse the inhibitory activity of the drug

Therapeutic application
Antiviral
Anticoagulant
Antiangiogenesis
Anti-inflammatory
Antiproliferation

RIBOZYMES
Ribozymes - RNA molecules capable of sequence specific

cleaving of mRNA molecules.

In the early 1980s,Cech and coworkers discovered the

selfsplicing activity of RNA enzymes of the group I intron

of Tetrahymena thermophilia
and coined the term
ribozymes

Two types of ribozymes, the hammerhead and hairpin

ribozymes, have been extensively studied for therapeutic

applications.
(Stull et al.,1995)

Hammerhead ribozy

Hairpin

Mechanism of
action of
Ribozymes

Delivery system dissociation

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Interaction with mRNA

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Activation of RNase H
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Inhibition of protein biosynthesis

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Inhibition of disease

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The presence of the RNA

backbone makes them easy

targets for degradation from
the ubiquitous RNases, so
these
molecules
are
biologically unstable in vivo.
(Merdan, 2000)
Stabilization of ribozymes is

more difficult than ASONs

Serum half-life of the stabilized ribozyme is increased to more than
10 days compared to a less than 1 min half-life of the unmodified RNA
ribozyme.

Therapeutic
application

ANGIOZYME is targeted against the vascular endothelial

growth factor (VEGF) receptor to reduce tumor growth by

inhibition of the angiogenesis.
(Wright and Kearney, 2001)

HEPTAZYME is another modified hammerhead ribozyme

that cleaves the internal ribosome entry site of the Hepatitis

C virus. The ribozyme was demonstrated to inhibit viral
replication up to 90% in cell culture.
(Macejak et al., 2001)

HERZYME is targeted against the human epidermal growth

factor-2 (HER2),which is overexpressed in certain breast

and ovarian cancers.
(Ribozyme Pharmaceuticals, USA)

DNAzymes
DNAzymes are analogs of ribozymes with

greater biological stability.

The RNA backbone chemistry is replaced by

the DNA motifs that confer improved

biological stability.

DNA sequences are also easy to modify synthetically,

thereby generating even stronger, resilient secondgeneration analogs

DNAzyme directed against the vascular endothelial

growth factor receptor 2 was confirmed to be capable of

tumor suppression by blocking angiogenesis upon
intratumoral injections in mice.
(Zhang et al ., 2002)

SiRNAs
SiRNAs
Short

double
stranded
RNA
segments of 21- to 23-nucleotide
bases
complementary
to
the
mRNA
sequence of the target protein.

First described for the nematode worm

Caenorhabditis elegans.

Mechanism of Action

Delivery system dissociation

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Formation of RNA-induced silencing

complexes
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Removal of one strand of single strand of

siRNA
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interaction with mRNA

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RISC induced degradation of mRNA

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inhibition of translation and protein

biosynthesis
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Specificity depends on the location of a particular

sequence within the mRNA.

(Gitlin et al., 2000)

dsRNAs longer than 30 nucleotides result in an

interferon response by cells, causing the dsRNA to bind

protein kinase R (PKR), which inhibits translation and
causes mRNA degradation by ribonuclease L.

To resolve this issue, short interfering RNAs (siRNA)

19-25 nucleotides in length are generally suggested.

(Elbashir et al., 2001)

Advantages
Their high degree of specificity to mRNAs,
Nonimmunogenic nature,
High resistance to ribonucleases.
Greater safety than plasmid molecules.
Require less sophisticated delivery systems,
Because of their small size, it possible to deliver a

cocktail of siRNAs targeting multiple disease-causing

genes in a single delivery system to control complex
diseases such as cancer where several genes are
malfunctioning.

Therapeutic application
Like Genasense (oblimerson), an siRNA against Bcl-2 was

used to treat malignant melanoma cells.

An anti-Bcl-2 siRNA
19-fold reduction in Bcl-2 mRNA
With low-dose cisplatin
complete inhibition of cell
growth (96%)
In cell lines and in primary cells from chronic myeloid
leukemia patients,
siRNA reduced expression of bcr-abl mRNA by 87%
shRNAs blocked the expression of bcr-abl protein by

more than 90%

ANTIGENE NUCLEIC ACID

COMPOUNDS
Nucleic acids targeted to genomic DNA

have been termed antigene nucleic

acids..
Gene specificity would be expected
only when the antigene compound
interferes with transcription processes.
Antigene compound bind with double
stranded DNA and form a triple helix
or triplex.

 Triplex formation
Prevent the interaction of various protein

factors required for transcription

Physically block the initiation or
elongation of the transcription complex.

Mechanism of Action
of
antigenic nucleic acids

Delivery system dissociation

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Entry into the Nucleus

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Interaction with host genome

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Inhibition of mRNA biosynthesis

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CELLULAR UPTAKE
The cellular uptake of naked DNA molecules

and polynucleotides
inefficient process.

remains

an

extremely

A combination of several inherent factors such

as DNA charge, size, and poor stability presents

a potent barrier to cellular uptake.

The negatively charged phosphate backbone of

the DNA molecule is the primary cause of its

inadequate and inefficient cellular association,
owing to electrostatic repulsion from the
negatively charged cell surface.

Basic of Cellular Uptake

DNA

molecules are internalized into the

cytoplasm by cell surface receptors via
receptor-mediated endocytosis.

Pinocytosis and absorptive endocytosis may

also play an important role in oligonucleotide

uptake.
Chemically

unmodified or naked DNA

therapeutics have extremely low in vivo
stability.

1. Mechanical and Electrical Techniques

Microinjection (micropipette/thin glass capillaries)
particle bombardment (Gold/tungsten particles)
use of pressure
electroporation.

Microinjection is highly efficient since one cell at a

time is targeted for DNA transfer.

(Luo et al., 2000)

2. Vector-Assisted Delivery
Systems
A) Viral Delivery Systems

For therapeutic purposes, the transgene of interest is

assembled in the viral genome and the virus uses its

innate mechanism of infection to enter the cell and release
the expression cassette. The gene then enters the nucleus,
is integrated into the host gene pool, and is eventually
expressed.

Retroviruses, parvoviruses, adenoviruses, lentiviruses,

adeno-associated viruses, and the herpes simplex virus are

being investigated for their ability to transfer DNA.

B) Nonviral Delivery Systems

Polymeric Delivery Systems
Polyethylenimine
Poly(L-lysine)
Dendrimers
Chitosan

Liposomal Delivery Systems

Cationic Liposomes.
Anionic Liposomes.
pH-Sensitive Liposomes.
Immunoliposomes.
Stealth Liposomes.

Advances in recombinant DNA technology and synthetic

chemistry have led to discovery of novel nucleic acid

drugs.

Major

improvements have been achieved by the

development of modified nucleotides that provide high
target affinity, enhanced biostability and low toxicity.

Most of the DNA-based drugs are in early stages of

clinical trials.

An Antisense oligonucleotide formulation, Vitravene and

an Aptamer formulation Macugen have received approval

from regulatory agencies.

By developing suitable drug delivery system, these drugs

can emerge as novel therapeutics in the area of antiviral,

anticancer and anti-inflammatory in near future.

Problems of stability and bioavailability should

be solved.
Methods of mass production and delivering

modified oligonucleotide should be developed.

THANK YOU