Antisense Drug Technology

Antisense is an innovative platform for drug discovery. Platform technologies combine all the
elements necessary to rapidly and efficiently create a stream of new products.

The targets for antisense drugs are RNA molecules. RNA is an optimal drug target for several
reasons:

 RNA is universal. It is vital to all living things, as it affects the expression of all genes.
 RNA is a simpler molecule than proteins in that it is composed of four fundamental
building blocks called nucleotides (A,C,U,G). Proteins are made from 20 different
building blocks called amino acids.
 RNA-‐based drug discovery is a rational and efficient process. We design our antisense
drugs to interact precisely with a specific sequence of RNA.

Antisense therapy is a form of treatment for genetic disorders or infections. When the
genetic sequence of a particular gene is known to be causative of a particular disease, it is
possible to synthesize a strand of nucleic acid(DNA, RNA or a chemical analogue) that will
bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively
turning that gene "off". This is because mRNA has to be single stranded for it to be
translated. Alternatively, the strand might be targeted to bind a splicing site on pre-‐mRNA
and modify the exon content of an mRNA.

This synthesized nucleic acid is termed an "anti-‐sense" oligonucleotide because its

base sequence is complementary to the gene's messenger RNA (mRNA), which is called the
"sense" sequence (so that a sense segment of mRNA " 5'-‐AAGGUC-‐3' " )

Antisense oligonucleotides have been researched as potential drugs for diseases such as
cancers (including lung cancer colorectal carcinoma, pancreatic carcinoma, malignant
glioma and malignant melanoma, diabetes Amyotrophic lateral sclerosis(ALS), Duchenne
muscular dystrophy and diseases such as asthma, arthritis and pouchitis with an
inflammatory component. As of 2014 two antisense drugs have been approved by the U.S.
Food and Drug Administration (FDA), fomivirsen (marketed as Vitravene) as a treatment
for cytomegalovirus retinitis and mipomersen (marketed as Kynamro) for homozygous
familial hypercholesterolemia.

1
Innovative Platform : Antisense Drug Discovery

Antisense is an innovative platform for drug discovery. Platform technologies combine all the
elements necessary to rapidly and efficiently create a stream of new products.

Cells are small membrane-enclosed compartments containing a host of structures and biochemicals
made of proteins or by the action of proteins. The genetic blueprint for the production and control of
these structures and biochemicals is found in the nucleus of the cell, where the chromosomes
contain about 30,000 genes, collectively called the human genome.
Each of our genes is a set of instructions for, and control of the manufacture inside the cell of a
unique protein. Some proteins form the cell structure, while others are enzymes that carry out the
functions of the cell or hormones interacting with the external environment of the cell.

Most pharmaceutical drugs available today interact with one or more proteins. They act to
enhance or inhibit the action of a protein or mimic its role thereby bringing about the
specified therapeutic effect.

To date the pharmaceutical drugs on the market target a total of only about 500 different
proteins. Although not all proteins will be suitable targets for therapeutic intervention, there
is clearly enormous scope for drug discovery and new therapies in this post genomic era,
considering that the human genome codes for at least 30,000 different proteins.

As technologies progress in the medical sciences, newer, more specific and rapid means of
drug discovery are emerging – antisense is one of these emerging technologies.
The Principle of Antisense Drug Technology :

Antisense technology prevents the production of proteins involved in disease processes,

which results in a therapeutic benefit to patients.

Proteins are fundamental components of all living cells and include many types of molecules,
such as enzymes, hormones and antibodies, necessary for carrying out the body's functions.
The overproduction or abnormal production of proteins is implicated or associated with
many diseases. Antisense prevents undesirable protein production in disease.

The antisense concept was first conceived in 1978 and has been through various technical
refinements. Antisense compounds are designed to have the right nucleotide sequence to bind
specifically to and interfere with its associated mRNA, the instructions for the production of a
particular protein. To create antisense drugs, special chemically stabilized nucleotides are
synthetically linked together in short chains of about 12-‐30 nucleotides (called
oligonucleotides). Each antisense drug is designed with the right complementary genetic code
to bind to a specific sequence of nucleotides in its mRNA target to form a short area of double
strands.

This double stranded region can inhibit the production of protein by a number of
mechanisms. They include stopping the ribosome from reading the message, or by leading to
the destruction of the mRNA by an enzyme already in the cells called RNase H. Rnase H
present both inside and outside of the nucleus, destroys any such double-‐stranded
nucleotides.

Antisense drugs are rapidly and effectively absorbed in the blood, antisense drugs bind
loosely to proteins. This binding facilitates their distribution to tissues and prevents
immediate loss in urine. In tissues, the drugs are cut by endonucleases, the drugs may be
further degraded by exonucleases. The partial drug molecules do not bind to proteins and are
therefore cleared in the urine.

Like all drugs, antisense drugs can have side effects. The side effects are generally predictable,
occur at high doses and are well understood. More than 6,000 humans have been safely
treated in trials conducted by Isis and its partners.

Antisense Approaches — Mechanisms and Targets :

An important area of basic research is to understand the molecular mechanisms of antisense

drugs. There are at least 12 known antisense mechanisms that can be exploited once an
antisense drug binds to its target RNA.

An antisense mechanism is defined as the process in which an antisense drug works after it
binds (hybridizes) to a target RNA forming a duplex. The formation of this duplex, or
two-‐ stranded molecule, prevents the RNA from functioning normally and from producing a
protein. The majority of late-‐stage antisense drugs in development bind to their target
RNA and activate a cellular enzyme called RNase H. Progress in research program is illustrated
by accomplishments in understanding RNase H.

There are more than a dozen antisense mechanisms that can be exploited with antisense
technology. While the majority of the drugs in pipeline bind to mRNAs and inhibit the
production of disease-‐causing proteins through the RNase H mechanism, it is believed
that antisense technology is broadly applicable to many different antisense mechanisms,
including RNA interference, or RNAi, and splicing, and many different RNA targets, including
microRNAs, long, non-‐coding RNAs and toxic RNAs. MicroRNAs are involved in the regulation
of protein production within the cell.

5
RNase H

The antisense mechanism that has been the main focus of research involves a cellular enzyme,
RNase H. This cellular enzyme is activated when antisense drugs bind to their target RNA and
activate RNase H. Upon activation, RNase H seeks out and destroys the target mRNA,
inhibiting a cell's production of a specific protein.

RNAi

RNAi is an antisense mechanism that involves using small interfering RNA, or siRNA, to target
a mRNA sequence. With siRNA, the cell utilizes a protein complex called RISC to destroy the
mRNA, thereby preventing the production of a disease-‐causing protein.

Alternative Splicing

To understand alternative splicing, it's important to understand how DNA creates mRNA.

mRNA is created when the entire DNA strand is copied. This includes the sequences that
encode for proteins and regions that are unnecessary for making proteins. Before mRNA can
function, the regions that are unnecessary for making proteins are deleted from the RNA
strand. The natural process that removes these regions and re-‐forms the finished mRNA
is called "splicing." Through the splicing process, the cell can create many diverse proteins
from a single gene and splicing accounts for most of the diversity in proteins in the cell. In
fact, of the approximately 25,000 genes in the human genome, approximately 90% have
alternative splice forms. Alternative splicing of proteins can result in the production of proteins
that are involved in disease. In some cases, using antisense technology, can direct alternate
splicing to produce a protein critical for normal cellular function to correct for a genetic defect.
Examples of applications of antisense modulation of splicing to treat genetic diseases include
spinal muscular atrophy (SMA), thalessemia, cystic fibrosis and Duchenne’s muscular
dystrophy.

MicroRNA

MicroRNAs are small naturally occurring RNA molecules that are created inside cells. There
are many different types of RNA that exist within the body, including mRNA. MicroRNAs are
important because they appear to have critical functions in controlling processes or pathways
of gene expression.

6
There are nearly 700 microRNAs that have been identified in the human genome, and these
are believed to regulate the expression of approximately one-‐third of all human genes.
Targeting microRNA to inhibit disease-‐causing pathways is an exciting development in RNA-‐
based therapeutics.

Types of Antisense Oligonucleotides :

First-generation antisense oligonucleotides

The first generation ODNs are synthesized by replacing one of the non-‐bridging oxygen atoms
in the phosphate group with either a sulfur group (phosphorothioates), methyl group (methyl
phosphonates) or amines (phosphoramidates). The first generation ODNs have more
resistance to nucleases and longer plasma half life as compared with phosphodiester
oligonucleotides. Moreover, they are easy to synthesize, carry negative charges that ease their
cell delivery, are capable of activating RNAse H and have suitable pharmacokinetics. At
present, the phosphorothioate is the most widely used As-‐ODN modification
and phosphorothioate oligonucleotides (PS-‐ODNs) have shown promising results both in vivo
and in vitro. The only FDA approved As-‐ODN drug, Vitravene, and most of the other
drugs in clinical trials are PS-‐ODNs.

However, as discussed to be later, first generation As-‐ODNs produce various undesirable, non-‐
specific in vivo side effects, such as immune stimulation and complement activation, which
are mainly caused by their interactions with proteins.

Second-generation antisense oligonucleotides

To improve the binding affinity and hybridization stability with target mRNA and to increase
the nuclease resistance, second generation As-‐ODNs, with alkyl modifications at the
2' position of the ribose, were developed. The most commonly used second generation As-‐
ODNs are 2'-‐O-‐Methyl (2'-‐OME) and 2'-‐O-‐Methoxyethyl (2'-‐MOE) ODNs. However, the useful
effects of these modifications are dampened by the fact that they render 2'-‐OME and
2'-‐MOE incapable of activating RNAse H, an endonuclease whose recruitment is considered to be
important for the activity of As-‐ODNs. To induce RNAse H activation, chimeric As-‐ODNs were

7
developed, by surrounding a central gap region consisting of a phosphorothioate deoxyribose
core, with nuclease resistant arms such as 2'-‐OME or 2'-‐MOE. The resulting “gapmer”
allows RNAse H to sit in the central gap to activate target specific mRNA degradation, while the
arms, by virtue of alkyl modifications, prevent the degradation of As-‐ODNs by nucleases.

Second generation As-‐ODNS are reported to have a higher affinity for mRNA, better
tissue uptake, increased resistance to nucleases, longer in vivo half life and lesser toxicity, as
compared to first generation As-‐ODNs. GEM 231 and GEM 92 (Hybridon) are examples
of second generation As-‐ODNs currently being tested in the clinical trials.

Third-generation antisense oligonucleotides

Third generation As-‐ODNs were developed by chemically modifying the furanose ring of the
ODNs, along with modifications of phosphate linkages or of riboses, as well as of nucleotides.
The modifications were made to improve the nuclease stability, target affinity and
pharmacokinetic profiles of the ODNs. Locked nucleic acid (LNA), Peptide nucleic acid (PNA)
and Morpholino phosphoroamidates (MF) are the three most commonly used third
generation As-‐ODNs.

Third generation As-‐ODNs have higher stability in biological fluids as they are
essentially resistant to degradation by nucleases and peptidases. They also exhibit a strong
hybridization affinity with the mRNA. In addition, PNAs, due to their ability to recognize double
stranded DNA, are capable of modulating gene expression or inducing mutation by strand
invasion of chromosomal duplex DNA. However, third generation As-‐ODNs do not activate
RNAse H and most likely produce their biological effects by causing steric hindrance of
ribosomal machinery, resulting in translational arrest. Moreover, being uncharged molecules,
they do not bind to serum proteins that normally bind poly anions. On the one hand, this
reduces the likelihood of non-‐specific interactions, but on the other hand, hastens their
clearance from the body. Also, their electrostatically neutral backbones make their solubility
and uptake difficult. To overcome these problems, in vitro, their delivery can be improved by
employing the delivery systems mentioned later in this chapter.

8
Therapeutic Applications

1. Potential Therapeutic Applications of Antisense Oligonucleotides in Ophthalmology

In the past several decades tremendous strides have been made toward understanding the
molecular basis of various ocular diseases. Modern day ocular pharmacology provides the
basis for utilization of rational drug design targeted toward molecular mechanisms in a
plethora of ocularconditions with the ultimate goal of developing effective therapeutics. The
application of anti-‐sense oligonucleotide (ASO) therapeutics for the treatment of
ocular diseases and conditions hastremendous opportunity to add treatment option in
ophthalmology. This class of pharmacologic

Classes of Therapeutic Oligonucleotides

Oligonucleotide therapeutic agents fall into two broad categories. The first includes oligonu-‐
cleotides that work through an antisense mechanism to inhibit the expression of a specific
protein.The inhibition of the production of a targeted protein involved in a disease process
results from thehybridization of an oligonucleotide to a single-‐stranded mRNA molecule and
activation of enzymesystems that subsequently degrade the mRNA, inhibit the translation
process, or modulate RNA processing. The single-‐stranded ASOs utilize the RNase H enzyme
mechanism and are furtherdivided into categories based on chemical modification as
described below. To date this first broadcategory is represented by fomivirsen sodium
(Vitravene, Isis Pharmaceuticals, Inc.) approved in theUnited States and Europe for the
treatment of cytomegalovirus (CMV) retinitis in patients with AIDS. The development of the
first antisense oligonucleotide to be approved is covered in moredetail in the section of this
chapter on antivirals. Also included in this category of antisense oligo-‐nucleotides are siRNA
inhibitors that function through the RISC-‐complex mechanism. The second broad category is a
class referred to as oligonucleotide aptamers which functionthrough the selection of an
oligonucleotide that will interact with a specific soluble protein or cell-‐surface receptor rather
than through hybridization with mRNA. In this case the interactionbetween aptamer and
target is dependent on the conformational structure of the oligonucleotide. The only approved
drug in this category is pegaptanib (Macugen, Pfizer, Inc.), a pharmacologic agent for the
treatment of neovascular age-‐related macular degeneration (AMD). Pegaptanib is a 28-‐base
ribonucleic acid aptamer covalently bound to polyethylene glycol, thatblocks the activity of the
vascular endothelial growth factor (VEGF), specifically VEGF165.The approval of pegaptanib, 6
years after the approval of fomivirsen, supports the use of oligo-‐nucleotides in the treatment of
retinal conditions through an intravitreous route of administration.While there are other
aptamers with the potential for use as ocular therapeutics, there are not manyexamples in this
category in the published literature. Therefore, this review will focus on the application of
antisense inhibitors.

9
2. Cardiovascular Therapeutic Applications
Coronary heart disease (CHD), despite tremendous medical and technological advances and
clearer insights into the pathogenesis of atherosclerosis, has been the leading cause of death in
the United States for over a century. Complications from atherosclerotic heart disease are the
most common causes of morbidity and mortality in industrialized nations. In fact, the World
Health Organization and others project that CHD will become the primary cause of death
worldwide by the year 2020. Several factors have been identified that increase the probability
that an individual might develop atherosclerosis. The major independent risk factors include
elevated total and low-‐density lipoprotein cholesterol (LDL-‐C) levels, low serum high-‐
density cholesterol (HDL-‐C) levels, cigarette smoking, hypertension, diabetes mellitus,
insulin resistance, and advancing age.Other factors include obesity, physical inactivity, family
history of premature cardiovascular disease, ethnicity, elevated serum triglyceride, homocysteine,
and lipoprotein(a) [Lp(a)] levels, and the presence of prothrombotic factors such as fibrinogen
and inflammatory markers such as C-‐reactive protein (CRP). These categories can be
further subdivided into those that can be modified through various interventions (alterations
in lifestyle and diet, or pharmacological agents) and those that cannot. Unmodifiable factors
include advancing age, gender, ethnicity, and genetic predisposition.Of those factors that can
be modulated, the abnormalities associated with lipid metabolism have been the most
extensively studied and therefore, the best understood.

3. Inflammatory Diseases
Antisense provides a novel pharmacological approach to the modification of the inflammatory
response. Advantages of antisense strategies include the ability to achieve hybridization-‐
based target specificity and the ability to reduce expression of a target protein in the nuclear,
cytoplasmic, ormembrane compartment(s) of cells. This combination of properties
distinguishes antisenseoligonucleotides (ASOs) from small-‐molecule and protein-‐
based technologies. In addition, thepharmacokinetic properties of first-‐ and second-‐
generation phosphorothioateoligodeoxyribo-‐nucleotides (PS-‐ODNs) are
compatible with more convenient, once
daily, or less frequent administration of antisense therapeutics in patients.Polygenetic
inflammatory diseases pose formidable challenges for antisense drug development, however,
including dose, dose interval, and delivery optimization. The involvement of aberrant localand
systemic host responses is apparent in syndromes such as Crohn’s disease, asthma, and
rheumatoid arthritis (RA). There is experimental evidence for heterogeneity in cell
responsiveness to ASOs,which may reflect differences in oligonucleotide uptake efficiency or
intracellular distribution.The evaluation of antisense therapeutics in inflammatory diseases
can also be complicated byoff-‐target proinflammatory effects of oligonucleotides. Both
first-‐ and second-‐generation PS-‐ODNsare capable of activating complement and
eliciting release of cytokines and chemokines. Cellsof the innate immune

10
 system may recognize internalized oligonucleotides via Toll-‐like receptor (TLR) 9.
The existence of non-‐TLR-‐based mechanisms for recognition of single-‐ and double-‐
stranded DNA have also been documented in human neutrophils and may also be involved in
theacute inflammatory response to specific sequence motifs. Second-‐generation, 2-‐O-‐
methoxyethyl (2-‐ MOE)-‐modified PS-‐ODNs have demonstrated increased pharmacological
potency relative tofirst-‐generation antisense inhibitors. Consequently, fewer
proinflammatory effects of 2-‐ MOEPS-‐ODNs are observed at clinically relevant doses.
Several lines of evidence can be pursuedto distinguish antisense effects of ODNs from
off-‐target effects in vivo, including demonstrations oftarget mRNA or protein reduction
concomitant with pharmacology and activity of multiple ASOsdirected to nonoverlapping target
mRNA hybridization sites. Mismatched oligonucleotides can alsobe evaluated in preclinical
studies to explore chemical-‐ class-‐related effects of ODNs. Collectively,these analyses
can provide insight into the mechanism(s) underlying oligonucleotide drug activity.While
significant challenges lie ahead for clinical development of antisense therapies forinflammatory
diseases, important achievements have recently been made. The clinical developmentstatus of
antisense therapies and the molecules that are likely to advance into the clinic for treatmentof
inflammatory diseases are the focus of this review.

4. Antisense Oligonucleotides for the Treatment of Cancer

Advances in tumor biology have identified many attractive molecular targets for new
drugdiscovery. The most promising candidates for antisense therapy are those targets that
become upregulated during and are causally related to cancer progression and therapeutic
resistance, and not otherwise amendable to inhibition with antibodies or small molecules.
Moreover, the target should be selectively overexpressed in tumor cells to minimize side
effects as tumor-‐selective uptake of ASOs cannot be ensured by standard protocols.
Although potential gene target libraries developed by microarray technology are valuable,
this information must be balanced by the inherent limitations of microarray analyses. These
include the inability to examine translational and posttranslational regulatory mechanisms
that impact the activity of various cellular proteins. In our laboratory,one method of identifying
potential therapeutic targets for prostate cancer starts with use of comparative hybridization
of high-‐density cDNA arrays to rapidly and efficiently characterize changes in gene expression
after androgen withdrawal in xenograft models that mimic the human condition of castration-‐
induced regression followed by androgen-‐independent progression. Computer-‐
assisted subtractive analyses of arrays highlight increases or decreases in gene expression at
various time points during progression. Northern or Western analysis is then used to confirm
the array data which can be verified in human tissue microarrays of untreated and
posthormone therapy-‐treated cancers.

11
Conclusion

The rapid development of antisense technology offers almost unlimited scope for the
development of new and highly specific therapeutics. Antisense Therapeutics therefore is well
positioned to play a significant role in the progression of antisense technology for drug
development in human diseases.

References

 http://en.wikipedia.org/wiki/Antisense_therapy

 http://www.antisense.com.au/technology-overview/

 Bennett, C.F.; Swayze, E.E. (2010). "RNA targeting therapeutics: molecular mechanisms
of antisense oligonucleotides as a therapeutic platform". Annu. Rev. Pharmacol. Toxicol.
50: 259–293.

 Pollack, Andrew (29 January 2013) F.D.A. Approves Genetic Drug to Treat Rare Disease
The New York Times.

 http://www.premedmag.org/2014/01/12/antisense-drugs/

 http://www.sciencedirect.com/science/article/pii/S0169409X15000101

12