See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/318084598

Antisense Technology,

Chapter · January 2012

CITATIONS READS

0 13,161

2 authors:

Sanjay Mohan Gupta Neha R Tomar

Defence Institute of Bio-Energy Research, DRDO, Haldwani, Uttarakhand , India Kumaun University
117 PUBLICATIONS   517 CITATIONS    13 PUBLICATIONS   48 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

TRANSCRIPTOME WIDE CHARACTERIZATION OF DOF TRANSCRIPTION FACTOR GENE FAMILY EXPRESSED IN LEAF AND DEVELOPING SPIKES OF FINGER MILLET FOR
DEFINING THEIR ROLE IN ACCUMULATION OF SEED STORAGE PROTEINS View project

All content following this page was uploaded by Neha R Tomar on 01 July 2017.

The user has requested enhancement of the downloaded file.

 Antisense Technology 297

CHAPTER

13 Antisense Technology

Antisense RNA and DNA techniques have been developed as a relatively recent approach to the
specific modulation of gene expression in vitro and in vivo. The purpose of this chapter is to lay
the foundation of basic concepts and practical uses of antisense technology in medical, veterinary,
agricultural sciences and industrial applications.

Keywords: Antisense technology, antisense-oligo-nucleotides, gene silencing, gene expression,

phosphorothioates.

INTRODUCTION
Antisense technologies are a suite of techniques that, together, form a very powerful tool for
studying gene function (functional genomics) and for discovering new and more specific treat-
ments of diseases in humans, animals and plants (antisense therapeutics). A conventional definition
of antisense refers to the laboratory manipulation and/or modification of DNA or RNA so that its
components (nucleotides) form a complementary copy of normal, or “sense,” messenger RNA
(mRNA). The binding or hybridization of antisense nucleic acid sequences to a specific mRNA
target will, through a number of different mechanisms, interrupt normal cellular processing of the
genetic message of a gene. This interruption, sometimes referred to as “knock-down” or “knock-
out” depending upon whether or not the message is either partially or completely eliminated, allows
researchers to determine the function of that gene. Three types of anti-mRNA strategies can be
distinguished. Firstly, the use of single-stranded antisense-oligonucleotides; secondly, the triggering
of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly,
RNA interference induced by small interfering RNA molecules (Figure 13.1). The overall goal in
introducing an antisense agent into cells either in vitro or in vivo is to suppress or completely block
the production of the gene product. This means that at some point in the transition from DNA
sequence to amino acid sequence, the normal transcription and translation apparatus must be
affected. Despite the seemingly simple idea to reduce translation by oligonucleotides complemen-
tary to an mRNA, several problems have to be overcome for successful application. Accessible
sites of the target RNA for oligonucleotide binding have to be identified, antisense agents have to
be protected against nucleolytic attack and their cellular uptake and correct intracellular localization
have to be achieved. In addition, RNA-cleaving ribozymes, deoxyribozymes and the use of 21-mer
double-stranded RNA molecules for RNA interference applications in mammalian cells offer highly
efficient strategies to suppress the expression of a specific gene. In this chapter, a survey of the
298 Biotechnology in Medicine and Agriculture

agents employed in antisense technologies will be presented along with a discussion of the various
mechanisms they employ to achieve the goal of reducing or eliminating normal processing of a gene
of interest.

Figure 13.1 Comparison of different antisense strategies. While most of the conventional drugs
bind to proteins, antisense molecules pair with their complementary target RNA.
Antisense-oligonucleotides block translation of the mRNA or induce its degradation
by RNase H, while ribozymes and DNA enzymes possess catalytic activity and
cleave their target RNA. RNA interference approaches are performed with siRNA
molecules that are bound by the RISC and induce degradation of the target mRNA
(Source: Kurreck, 2003).

Among several possible strategies for inactivating a single, chosen gene, the most approved one
is antisense technology. This modification does not involve actual subtraction but inactivation of
gene or suppressing gene activity. When the DNA strand is normally transcribed, the RNA pro-
duced is called sense RNA, complementary to DNA, but if the orientation of gene to be transcribed
is reversed with respect to promoter, the RNA transcribed from it would be reversed too, and the
RNA produced so has sequence same to that of antisense strand of the normal gene, thus known
as antisense RNA (asRNA). The basis of antisense technology is the use of antisense RNA. The
gene to be cloned is ligated into the vector in reverse orientation. Thus on transcription, the RNA
synthesized is reverse complement of the mRNA transcribed from normal version of gene, and
being complementary to each other, they will pair to form a hybrid. Thus this makes mRNA
unavailable for translation and the resulting double-stranded RNA molecule is degraded by specific
ribonucleases.
Antisense refers to short DNA or RNA sequences, termed oligonucleotides, which are designed
to be complementary to a specific gene sequence. The goal is to alter specific gene expression
 Antisense Technology 299

resulting from the binding of the antisense oligonucleotide to unique gene sequences. The antisense
effect of a synthetic oligonucleotide sequence was first demonstrated in the late 1970s by Zamecnik
and Stephenson (1978). Using nucleotide sequences from the 5¢ and 3¢ ends of the 35S RNA of
Rous sarcoma virus (RSV), Zamecnik and Stephenson identified a repeated sequence of 21 nucle-
otides (nt) that appeared to be crucial to viral integration. They correctly concluded that the
oligonucleotide was inhibiting viral integration by hybridizing to the crucial sequences and blocking
them. Simultaneous other groups, notably Tennant et al. (1973) and Miller et al. (1977) also
reported similar effects for synthetic oligonucleotides in other systems. Moreover, Matsukura and
colleagues demonstrated that phosphorothioated oligonucleotides were effective hybridons against
HIV replication in cultured cells.
The impact of biotechnology on antisense technology is expected to increase dramatically as the
links between genetics, protein production and disease are better understood. Currently, antisense
technology is used to design therapeutic compounds, which target specific mRNA sequences to
obstruct the production of certain disease-causing proteins. Traditional drug therapies focus on a
drug’s interaction with the disease-causing proteins. However, antisense drug therapies inhibit the
production of the disease-causing proteins altogether. Applications of antisense technology in
therapeutics are expected to increase substantially as the link between diseases and genes are being
discovered. Antisense drug therapies are currently unavailable in Canada. However, there are
numerous antisense therapies in development, some of which are in clinical trials, and one of which
has been approved for use in the United States.

CONCEPTS
Clearly, the antisense concept derives from an understanding of nucleic acid structure and function
and depends on Watson-Crick hybridization. Thus, arguably, the demonstration that nucleic acid
hybridization is feasible and the advances in in situ hybridization and diagnostic probe technology
laid the most basic elements of foundation supporting the antisense concept.
However, the first clear enunciation of the concept of exploiting antisense oligonucleotides as
therapeutic agents was in the work of Zameenik and Stephenson in 1978. They had synthesized
an oligodeoxyribonuleotide, 13 nucleotides long, which was complementary to a sequence in the
respiratory syncytial virus genome. They suggested that this oligonucleotide could be stabilized by
3¢ and 5¢ terminal modifications and showed evidence of antiviral activity. More importantly, they
discussed possible sites for binding in RNA and mechanisms of action of oligonucleotide.
Though less precisely focused on the therapeutic potential of antisense oligonucleotides, the
work of Miller and their collaborators during the same period helped establish the foundation for
antisense research and reestablish an interest in phosphate backbone modifications as approaches
to improve the properties of oligonucleotides. Their focus on methyl phosphotriester-modified
oligonucleotides as a potential medicinal chemical solution to pharmacokinetic limitations of oligo-
nucleotides presages a good bit of the medicinal chemistry to be performed on oligonucleotides.
Despite the observations of Miller and Zameenik, interest in antisense research was quite limited
until the late 1980s when advances in several areas provided technical solutions to a number of
impediments. As antisense drug design requires an understanding of the sequence of the RNA
target, the explosive growth in the availability of viral and human genomic sequences provided the
300 Biotechnology in Medicine and Agriculture

information from which “receptor sequences” could be selected. The development of method for
synthesis of research quantities of oligonucleotide drugs then supported antisense experiments on
both phosphodiester and modified oligonucleotides. The inception of the third key component
(medicinal chemistry) forming the foundation of oligonucleotide therapeutics, in fact, is the syn-
thesis in 1969 of phosphorothioate poly rI; poly rC as a mean of stabilizing the polynucleotide.
Subsequently, Miller and Ts’o initiated studies on the neutral phosphate analogs,
methylphosphonates, and groups at the National Institute of Health and the Food and Drug Admin-
istration and the Worcester Foundation investigated phosphorothionate oligonucleotides. With these
advances forming the foundation for oligonucleotide therapeutics and the initial studies suggesting
in vitro activities against a number of viral and mammalian targets, interest in oligonucleotide
therapeutic intensified.

PRINCIPLES
In principle, antisense technology is supposed to prevent protein production from a targeted gene.
The exact mechanism by which this occurs remains uncertain. Proposed mechanisms include
triplex formation, blocking RNA splicing, preventing transport of the mRNA antisense complex into
the cytoplasm, increasing RNA degradation, or blocking the initiation of translation (Figure 13.2).
Initially, cellular nucleases dramatically reduced the effectiveness of antisense oligonucleotides by
rapidly degrading these molecules after administration. These obstacles can be overcome in appli-
cations utilizing synthetic oligonucleotides by altering the nature of the phosphodiester bond by
replacing oxygen with sulfur. Such modified oligonucleotides are termed phosphorothionates.
Delivery of antisense oligonucleotides into target cells or the cell nucleus has been problematic. The
variety of viral and non-viral delivery systems previously discussed is currently being explored to
overcome this obstacle. Animals treated with antisense oligonucleotides have had significant side
effects, some of which have been lethal. Currently, the most problematic aspect associated with
antisense technology revolves around the specificity of their action. In some cases, non-specific

Figure 13.2 Principles of antisense technology (Source: www.deakin.edu.au/... 10857_mru_

n_graph5.gif).
 Antisense Technology 301

antisense sequences, in other words, sequences which do not bind to the targeted gene or RNA,
have prevented gene expression to the same degree as their sequence-specific antisense counter-
parts. This has led to considerable complication in data interpretation and requires detailed and
careful data analysis before contemplation of clinical trials. Since antisense technology focuses on
preventing gene expression, it has been most widely applied in cancer gene therapy.

ANTISENSE TECHNOLOGY
Antisense technology was first effectively used in plants to alter the levels of various degradative
enzymes or plant pigments. The technology was rapidly applied to mammalian cells and in 1992
Science named antisense its runner-up in the molecule of the year award.

What is Antisense Technology?

In antisense technology, synthetically-produced complementary molecules seek out and bind to
messenger RNA, blocking 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 protein
production. By binding to mRNA, the antisense drugs interrupt and inhibit the production of
specific disease-related proteins. “Sense” refers to the original sequence of the DNA or RNA
molecule. “Antisense” refers to the complementary sequence of the DNA or RNA molecule.

How Does Antisense Technology Work?

While traditional drug therapies are based on designing compounds that block or inhibit disease-
causing proteins, antisense therapies focus on preventing the production of disease-causing pro-
teins. Antisense drugs are based on small DNA-like or RNA-like constructs that bind to the protein-
coding strand of genetic message (mRNA), blocking the translation of the disease-causing protein.
By binding to mRNA, antisense drugs prevent the genetic code from being read by the ribosome,
which is responsible for translating and manufacturing proteins. Additionally, the bound antisense/
mRNA complex is enzymatically degraded so the protein cannot be synthesized (Figure 13.3).

Figure 13.3 Antisense therapy blocks the translation phase of proteins synthesis by binding to
the specific genetic segment of mRNA that code for production of a specific dis-
ease-causing protein. OGX-011 binds to the segment of mRNA that code for
clusterin (Source: employees.csbsju.edu/.../bind/antisense.GIF).
302 Biotechnology in Medicine and Agriculture

Evolution of Antisense Technology

The first researchers who worked on antisense technology saw the possibilities of new methods
of drug design. However, that promise has not yet been fully realized. First-generation drugs have
demanded daily, intravenous dosing, and their lower specificity for their target mRNA keeps them
from being as potent as is desirable. New developments in the chemistry of antisense give further
hope for developing potent, long-lasting antisense therapies that are administered more conve-
niently. The first antisense constructs studied were based on the natural backbone structure of
DNA. These constructs had an extremely high affinity for their target sequences. However,
structures based on naturally occurring DNA are subject to swift degradation by nucleases and
proved to be unsuitable for therapeutic uses. In an effort to make antisense technology more
amenable to therapeutic use, scientists attempted to alter the backbone of the antisense molecules.
The first successful backbone chemistry modified the phosphodiester structure in the DNA back-
bone to a phosphorothioate structure, which increased the construct’s stability and made the
technology more amenable to therapeutic use. However, these constructs still had plasma half lives
of only hours, and the modified DNA backbone decreased the affinity of the antisense molecule
to its target mRNA sequence. To increase the stability of the antisense compound, a second-
generation chemistry was developed, adding the 2’MOE (2’-methoxyethyl) modification to the
oligonucelotide backbone. A further improvement incorporates both first-generation and second-
generation chemistries into “gapmers”: antisense sequences that have their ends modified with
2’MOE chemistry while retaining at their centers first-generation chemistry. The 2’MOE modified
ends protect the construct from degradation, giving significantly improved half life and binding
affinity, while the first generation center permits enzymatic degradation of the mRNA/antisense
complex. Gapmer second-generation chemistry drugs have increased half-lives of 5- to 10-fold
over first-generation chemistry and increased affinity to target mRNA. The improved affinity of
second-generation drugs is primarily attributable to their design and composition. Second-genera-
tion drugs are composed of both RNA-like and DNA-like nucleotides, while first-generation drugs
are entirely DNA-like. Since RNA hybridizes more tightly to RNA than DNA, the second-generation
drugs have a greater affinity for their RNA targets and, therefore, greater potency. With increased
potency, second-generation drugs are more active at lower doses. Additionally, second-generation
chemistry with gapmer design significantly slows degradation of the drugs by protecting the drug
from destructive nucleases (Figure 13.4). The resulting slower clearance from the body allows for
less frequent dosing, and in the future may allow for oral delivery—a useful feature for long-term
use given added patient convenience.

Antisense Oligonucleotide Design

Biognostik offers a worldwide unique Antisense Custom Design Service, which selects optimized
antisense sequences against any target mRNA of interest. Biognostik has developed a proprietary
sequence design technology called .R.A.D.A.R.Ò, which allows rational selection of high quality
antisense oligonucleotides that meet the following criteria: highly specific, functional via a true
antisense mechanism, absence of non-specific effects, no protein binding, good cellular uptake
 Antisense Technology 303

Figure 13.4 Evolution of synthetic antisense oligonucleotides (Source: Crooke, 2004).

pattern and stabilization by phosphorothioate backbone modifications. R.A.D.A.R.Ò design com-

bines computer aided sequence analysis, molecular modeling and comparison to a core data base
in which analysis data of over 3000 antisense sequences are archived.

APPLICATIONS OF ANTISENSE TECHNOLOGY

Antisense technology has potential applications in medical, veterinary, agricultural sciences and
industries. Some of the important applications of antisense technology are discussed below.

Medical Applications
Diseases are often connected to the insufficient or excess production of certain proteins. If the
production of these proteins is disrupted, many diseases can be treated or cured. Antisense
technology is a method that can disrupt protein production. It may be used to design new thera-
peutics for diseases in whose pathology the production of a specific protein plays a crucial role.
Antisense technology may play a major role in cancer chemotherapy. It is clearly a tool of
exceptional value in the fictionalization of genes and their validation as potential targets for cancer
therapy. Additionally, there is now substantial voidance that antisense drugs are safe, and a growing
body of data showing activity to animal models of human diseases including cancer and suggesting
efficacy in patients with cancer. Antisense technology is arguably the most advanced genomically
based drug discovery technology. It has been shown to be capable of generating very specific
inhibitors, and a significant number of antisense drugs are in development as anticancer agents.
304 Biotechnology in Medicine and Agriculture

Figure 13.5 Pre-mRNA is transcribed from a gene. It is then processed through a carefully
choreographed set of steps to the mature mRNA which is exported to the cytoplasm.
These steps include 5' and 3' modifications, (5' cap: 3' poly A) splicing and specific
transport activities. Numerous mechanisms of action for antisense drugs have
been identified and they affect many steps post transcription and/or enhance cellu-
lar degradation of the RNA (Source: Kurreck, 2003).

A number of potential mechanisms by which antisense drugs may work have been identified
(Figure 13.5) and they have been partitioned into two broad groups: occupancy only and occu-
pancy activated destabilization. Of the occupancy only mechanism, translation arrest and inhibition
of splicing have been most extensively characterized. Occupancy-induced destabilization, as the
target RNA, is epitomized by RNaseH degradation of the target RNA, and this mechanism is the
most often used and least characterized antisense mechanism.
There are now a number of reports of antisense inhibition of human tumour zenograft and other
animal models of human cancers. For many of these studies, appropriate controls, including a
variety of mismatched oligonucleotides were used and there is evidence that the effect of the
antisense agents were indeed due to an antisense mechanism. Human tumor zenografts have shown
that it is feasible to reduce target RNA and protein levels and to induce antiproliferative effects in
some of these models. Similar data has been reported by other groups.
Tamoxifen (TAM) is widely used in the treatment and prevention of breast cancer. There is a
clear evidence that cytochrome P450 (CYP) 3A enzymes play an important role in TAM metabo-
lism, resulting in metabolites that lead to the formation of TAM–DNA adducts. It has been reported
that the effect of CYP3A2 antisense (AVI-4472) exposure on CYP3A2 transcription, enzyme
activity, translation, and TAM–DNA adducts, in livers of rats resulted in down-regulation of several
CYP genes, suggesting the utility of antisense technology in the redirection of TAM metabolism.
 Antisense Technology 305

This approach of ‘‘metabolic redirection’’ builds on literature reports implicating the metabolic
products of the CYP3A subfamily of enzymes in the genotoxicity of TAM. The utility of antisense
phosphorodiamidate morpholino oligomers (PMO) in inhibiting the major CYP3A isoforms in rats
and humans has been demonstrated previously.
In the past decade, substantial strides have been made in creating and validating antisense
technology. There are several antisense drugs in clinical development that have shown some
promise, but only added clinical trials will determine their true value. New generation of antisense
drugs are in development and these may offer even more benefits to patients with cancer. If these
can be coupled to better target focused decision making deriving from antisense target validation,
perhaps even more benefit can accrue. Finally, the therapeutic index of antisense drugs supports
their use in combination with traditional cytotoxics drugs or, potentially more exciting, the com-
bination of antisense inhibitors with several different genes.
Antisense oligodeoxynucleotides (ODNs) inhibit target gene expression and are applicable to
human diseases including malignancies. Although improvements in ODNs and cellular delivery
systems are needed, antisense ODNs hold great promise as a new class of therapeutic agents.
Blocking the expression of specific genes with antisense oligonucleotide (ODNs) may promote new
therapies for many human diseases. The antisense ODN is usually designed as short (about 20 bp)
single-stranded DNA directed to the mRNA of the target gene. Binding to a complementary mRNA
sequence followed by prevention of its translation is a basic principle in this strategy. However, a
number of mechanisms of antisense ODN actions can produce target gene nonspecific-effects. For
example, the sequence can bind to DNA in the nucleus and block the transcription or interact with
small molecules and proteins, including the Sp1 transcription factor leading to its activation. Such
nonspecific-effects limit the conclusion that can be drawn from the results of the experiments using
antisense ODNs. However, gene-specific effects were shown in in vitro experiments and animal
models. Moreover, clinical trials have shown no remarkable toxicity and suggested therapeutic
effects of antisense ODNs in Crohn’s disease and malignant lymphoma. Thus, use of antisense
ODNs may be still fascinating to explore as a way to treat viral infection, prevent organ allograft
rejection, and eradicate malignancies.

Veterinary Applications
Antisense technology has potential applications in veterinary medicine and pharmaceuticals.
Transgenic technologies are used for improving milk production and the meat in farm animals as
well as for creating models of human diseases. Transgenic animals are used for the production of
proteins for medical use. Biotechnology is applied to facilitate xenotransplantation from animals to
humans. Genetic engineering is done in farm animals and nuclear transfer technology has become
an important and preferred method for cloning animals.
Antisense technology has potential applications in the management of several animal diseases
such as foot-and-mouth disease, classical swine fever, avian flu and bovine spongiform encepha-
lopathy. The most important biotechnology-based products consist of vaccines, particularly geneti-
cally engineered or DNA vaccines. Gene therapy for diseases of pet animals is a fast developing
area because many of the technologies used in clinical trials humans were developed in animals and
many of the diseases of cats and dogs are similar to those in humans. RNA interference technology
is now being applied for research in veterinary medicine. Molecular diagnosis is assuming an
306 Biotechnology in Medicine and Agriculture

important place in veterinary practice. Polymerase chain reaction and its modifications are consid-
ered to be important. Fluorescent in situ hybridization and enzyme-linked immunosorbent assays
are also widely used. Newer biochip-based technologies and biosensors are also finding their way
in veterinary diagnostics. Biotechnology products are approved by the Center for Veterinary Medi-
cine of the FDA. Regulatory issues relevant to animal biotechnology are described. Approximately
108 companies have been identified to be involved in animal biotechnology and are profiled in the
report. These are a mix of animal healthcare and biotechnology companies. Share of biotechnology-
based products and services in 2007 is analyzed and the market is projected to 2017.

Agricultural Applications
Genetic manipulations of fruit ripening
Controlling the rate of ripening in climacteric fruits is of prime importance in increasing their shelf
life. The ripening process in climacteric fruits could not be controlled efficiently by various means
such as modern storage facility, cool temperature storage, chemical sprays etc. once the ripening
has been initiated. Therefore, molecular genetic approach such as antisense technology could be
an ideal approach to control the ripening in climacteric fruits even after the ripening has set in.
Ripening is a highly complex final phase of fruit development that involves the expression and
regulation of hundreds of genes that can be used for manipulating fruit ripening. Some of the
important genes are discussed below.
Genes related to ethylene biosynthesis: Small multi-gene families code for the ACS and ACO
enzyme involved in the ethylene biosynthesis. It was observed that tomato fruits of transgenic plant
carrying ACS in antisense orientation completely blocked the ripening by decreasing ethylene
production to 99%, and these fruits did not ripen without exogenous ethylene. Similarly, anti-ACS
apple showed reduced autocatalytic ethylene production and increased shelf life. Tomato fruits
from transgenic tomato plants carrying tomato ACO in antisense orientation and melon fruits from
transgenic melon plants carrying ACO from melon under the regulation of CaMV35S promoter
showed a reduction in ethylene production and delayed ripening.
Genes related to cell wall softening: Several enzymes like polygalacturonase (PG), PME,
expansin, cellulose, xyloglucanase etc. are implicated in wall softening during ripening. Transgenic
homozygous tomato plants carrying PG gene in antisense orientation showed a reduced PG activity
1% of the normal value. It has been shown that in fruits with antisense PG, the degradation of
cellular wall pectins was inhibited (Figure 13.6) but other aspects of maturation, such as ethylene
production and lycopene accumulation were not affected. Ripened fruits from transgenic straw-
berry plants carrying pectate lyase from strawberry in antisense orientation under the expression
of CaMV35S were significantly firmer than control fruits and this gene could be an excellent
candidate for biotechnological improvement of fruit softening in strawberry. Suppression of Exp1
in tomato lead to firmer fruits as compared to wild type and also substantially inhibited polyuronide
depolymerization late in ripening, but did not prevent the breakdown of structurally important
hemicelluloses, a major contributor to softening. Antisense suppression of deoxyhypusine synthase
in tomato delays fruit softening and alters growth and development. Transgenic strawberry plants
carrying antisense pectate lyase cDNA from strawberry under the control of the 35S promoter
 Antisense Technology 307

showed significantly firmer ripened fruits than controls. Highest reduction of softening in these
fruits occurred during the transition from the white to the red stage. The postharvest softening of
apple fruit was also diminished.

Figure 13.6 Antisense polygalacturonase transgenic tomato (left panel) and wild type (right
panel) (Smith et al., 1988).

Genes related to lycopene production: Lycopene possesses beneficial antioxidant properties due
to which the researchers aimed to increase its content. Transgenic approaches were employed to
increase tomato fruit lycopene content. Suppression of lycopene cyclase (Lcy) gene resulted in
increased lycopene content in the transgenic tomato fruits. Suppression of TDET1 (Tomato DE-
ETIOLATED1) mRNA accumulation by RNA interference (RNAi) specifically in fruits using
TDET1-derived inverted-repeat constructs driven by three different fruit-specific promoters, P119,
2A11 and TFM7 was shown to enhance the carotenoid and flavonoid content in mature fruit.

Modification of flower colour in decorative plants

Antisense technology can be used for producing decorative plants exhibiting one or more desired
phenotypic traits by manipulating genes involved in flavonoid biosynthetic pathway (Figure 13.7a).
The class of genes within the flavonoid biosynthetic pathway includes the nucleic acid sequences
involved directly in reactions or control of reactions, which synthesize or modify a flavonoid
compound whose functions in plants include coloration in flowers, fruits, leaves, and other organs.
Examples of flavonoid biosynthetic genes include those for chalcone synthases, chalcone
isomerases (CHI), flavanone 3-hydroxylases, dihydroflavonol reductases, flavanone 2-hydroxy-
lases, dihydroflavanol 2-hydroxylases, flavonoid 3'-hydroxylases, flavonoid 5'-hydroxylases, fla-
vonoid glycosyltransferases (including glucosyl transferases such as UDPG: flavonoid 3-O-
glucosyl transferase and UDPG: flavonol 7-O-glucosyl transferase, and rhamnosyl transferases),
flavonoid methyltransferases (such as SAM:anthocyanidin 3-(p-coumaroyl)-rutinoside-5-glucoside
3',5'-O-methyltransferase) and flavonoid acyltransferases. The biosynthetic pathways of these
308 Biotechnology in Medicine and Agriculture

various pigments have been extensively studied in many different plant species. The chalcones and
aurones are products requiring only the initial biosynthetic enzymes, being direct products of the
earliest precursors. The flavones and flavonols are intermediate, and the anthocyanins are products
requiring substantial modifications from the initial precursors. All of these products are dependent
upon the activity of the initial enzyme, chalcone synthase (CHS), which catalyses the production
of chalcone from three molecules of malonyl-coenzyme A and one molecule of coumaroyl-coen-
zyme A.

(A) (B)
Figure 13.7 (A) Petunia plants in which genes for pigmentation are silenced. The left plant is
wild-type; the right plants contain transgenes that induce suppression of both
transgene and endogenous gene expression, giving rise to the unpigmented white
areas of the flower. (B) Modified Rosa x hybrida using a gene encoding chalcone
synthase to alter the anthocyanin biosynthetic pathway (Source: Souq et al., 1996).

In an effort to search for new flower colour, modified Rosa x hybrida using a gene encoding
chalcone synthase to alter the anthocyanin biosynthetic pathway (Figure 13.7b). Enhanced resis-
tance to powdery mildew was observed in GM Rosa x hybrid cv. Carefree Beauty modified with
an antimicrobial protein gene. Genetically modified Rosa x hybrida rootstock cv. Moneyway
containing rol genes from A. rhizogenes showed a threefold improvement in adventitious rooting
of cuttings. Recent work on cloning and expression of 1-aminocyclopropane-1-caroxylate syn-
thase from Rosa may lead to regulation of ethylene response in plants and potentially a longer vase
life for roses, in which ethylene is a hormonal regulator of senescence of most plant organs.

Antisense SAHH gene for resistance against a broad spectrum of viruses in plants
Antisense SAHH gene was used, which conferred resistance against one specific virus. However,
if we could inhibit the expression of a gene responsible for a non-essential host cell enzyme required
for virus replication, we can get resistance against a broad spectrum of viruses. In one such effort,
the enzyme S-adenosyl homocysteine hydrolase (SAUH) was chosen for manipulation. SAHH is
responsible for several methylation reactions including 5'-cap mRNA methylation, where S-
adenosyl-methionine is used as a methyl donor. If SAHH is inhibited, there will be under-methylation
at the 5' end of viral mRNA leading to inhibition of viral replication. SAHH inhibitions have actually
been used as antiviral agents and were found to suppress plant viruses in leaf disk assays. In a study
reported in 1995, transgenic tobacco plants were produced which expressed antisense RNA against
mRNA of a tobacco SAHH gene. This led to the reduction in SAHH level and consequent inhibition
of viral replication/infection in the transgenic plants.
 Antisense Technology 309

Mutant gene for a protein necessary for cell to cell movement of virus in the host plant
In certain viruses (e.g., white colour mosaic virus or WCIMV), there are genes that encode non-
virion proteins necessary for cell to cell movement of the virus (but not necessary for viral
replication). A mutation in one of these genes may hamper cell to cell movement of the virus. One
such approach was used (reported in 1994), where a mutant version of a gene encoding WCIMV-
13kDa protein was used for producing transgenic plants. The transgenic plants were found to be
resistant to several viruses. The mutant transgene was found to be dominant over the normal gene
of virus infecting the transgenic host.

Industrial Applications
Antisense is an important technology for drug discovery and development (Figure 13.8). It is
broadly used by the pharmaceutical industry as a tool for functional genomics and as highly specific
drugs for a wide range of diseases. Cumulative clinical and preclinical data suggest that antisense
technology has the potential to create a new sector of the pharmaceutical industry. After 20 years
of technology refinement, antisense is a mature drug discovery and therapeutic platform. Several

Figure 13.8 Strategies for the development of antisense drug (Source: www.antisense. com).
310 Biotechnology in Medicine and Agriculture

antisense drugs are now in late stage clinical development with one antisense drug developed by
Isis, Vitravene®, marketed in the US and Europe. Antisense inhibitors can target specific aspects
of ocular disease processes and have ideal properties as therapies for the eye. Antisense drugs have
already been shown to be effective in treating ocular diseases with the commercialization of
Vitravene®, approved for the treatment of CMV Retinitis.
With access to Isis Pharmaceuticals’ proprietary drug discovery process as illustrated in the
Figure 13.8, Antisense Therapeutics Limited can move quickly from drug discovery into developing
therapies. Once we have identified a therapeutic application and corresponding gene target, an
antisense lead inhibitor compound can be rationally designed within hours suitable for use in
research and clinical trials. This compares with traditional drug discovery approaches that can take
years to produce such a lead compound. Antisense drug development also benefits from uniform
methods of manufacture, formulation and delivery of antisense compounds. At the same time, the
explosion of genetic information provided by the completion of the Human Genome Project (HGP)
provides a rich resource of information on target genes for the design of antisense drugs. Vitravene
is a registered trademark of Novartis AG.
Isis scientists have worked systematically and rigorously to address the questions associated
with the development of this new drug discovery platform. Do antisense drugs interact with their
RNA targets in a highly specific manner? Where do antisense drugs distribute in the body? Do they
get into cells? What doses produce therapeutic effects? What are the potential toxicities, and how
can they be minimized or avoided? How can antisense drugs be made more convenient for patients?
What diseases can they treat?
Based on extensive research conducted to answer these questions, Isis scientists know that
antisense drugs possess the following characteristics:
Specificity: The pharmaceutical industry’s primary quest in drug discovery is drug specificity.
The more precisely a drug interacts with its intended target, and not with unintended molecules,
the more likely it is to produce a therapeutic effect without causing unwanted side-effects. In
contrast, traditional drugs bind based on the shape of proteins and charge interactions, creating
more opportunity for unwanted interactions, and, often undesirable side-effects.
Broadly applicable: The nature of the antisense receptor, RNA, makes virtually any molecular
target accessible to antisense drugs. There are many highly desirable molecular targets for drug
discovery that are considered “undruggable” by traditional small molecule technology. These
targets are often members of families of closely related proteins that are too similar in structure
for traditional drugs to distinguish amongst, the biological function of the protein is unknown, or
it is difficult to develop an assay for drug screening. Antisense drugs discriminate between targets
based on their genetic sequence, so the universe of potential targets is significantly greater.
Rational design: Antisense drug discovery is more rational than any other type of drug discovery
because: (1) the rules for creating these drugs are known, as they bind by hybridization to the target
RNA; (2) the chemistry is constant, with only the order of the drug’s nucleotides changing to make
the drug target specific; and (3) the distribution and metabolism of antisense drugs are very similar
from drug to drug, resulting in a common and often times, predictable, safety profile across
antisense drugs.
 Antisense Technology 311

Efficiency: The benefit of a more rational approach to drug discovery is efficiency. The effi-
ciency of antisense results in shortened timelines to identify lead drug candidates in the discovery
phase of development. The consistency of the behavior of antisense drugs allows Isis scientists
to apply what they learn from the testing of one drug to future drugs, thus reducing the potential
for failure in the early stages of development and creating significant competitive advantage for the
platform.

Redirection of drug metabolism using antisense technology

The cytochrome P450 (CYP) family is the most catalytically versatile component of the phase I
oxidation metabolic pathway and participates in the metabolism of a large majority of drugs used
in clinical practice. The inhibition of specific enzymes of this family can significantly alter the
disposition and toxicity of substrate drugs by reducing and/or redirecting their metabolism. It is
reported that the approaches available for CYP inhibition, with particular emphasis on the potential
use of antisense phosphorodiamidate morpholino oligonucleotide strategies to inhibit human
CYP3A4. Inter-individual variations of 10- to 50-fold have been reported in the activity of CYP3A4
enzyme, which contributes to the metabolism of more than half of all clinically relevant drugs. The
application of antisense technology for inhibition of specific CYP enzymes can provide significant
therapeutic benefits, including: (i) reduction of first-pass drug metabolism; (ii) reduction in drug
dosage; (iii) selective reduction of toxic metabolites; and (iv) increased oral/topical drug
bioavailability. The use of antisense morpholino oligonucleotide strategies to target CYP enzymes
may result in safer and more uniform therapeutic applications.
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, consid-
ering 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 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.

CONCLUSION
Antisense technologies have been hailed as one of the best options for gene manipulation to be used
in studying gene function and for discovering new and more specific treatments for a wide range
of diseases in humans, animals, and plants. However, this success has been mainly restricted to
laboratory and research organizations. There has been only one antisense drug in the market, even
after almost three decades of research on antisense technologies. Its clinical applications thus far
were barred by numerous problems and untrammeled challenges. Despite all the challenges,
antisense therapy has held its ground for more than two decades and now it is ready to emerge
312 Biotechnology in Medicine and Agriculture

as a potential tool for gene therapy. With more than a dozen molecules in the clinical development
phases, the stage is set for antisense therapy to emerge as a potential option for treatment of a wide
range of diseases. The antisense strategy of inhibiting gene expression has tremendous potential in
the fields of functional genomics, drug discovery and therapy.

FURTHER READINGS
1. Agrawal S., Goodchild J., Civeira M.P., Thornton A.H., Sarin P.S. and Zamecnik P.C. (1988) Oligo-
deoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency
virus. Proc. Natl. Acad. Sci. USA. 85 (19): 7079-7083.
2. Alvarado-Urbina G., Sarhe G.M., Liu W.C., Giller M.F, Duck P.D., Bender R. and Ogilvie K.K. (1981)
Automated synthesis of gene fragments. Science. 214 (4518): 270-274.
3. Arora V. and Iversen P.L. (2001) Redirection of drug metabolism using antisense technology. Current
Opinion in Molecular Therapeutics. (3): 249-257.
4. Ayub R., Guis M., Ben Amor M., Gillot L., Roustan J.P., Latche A, Bouzayen M. and Pech J.C. (1996)
Expression of A.C.C. oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nat. Biotechnol.
(14): 862-866.
5. Baker B and Monia B.P. (1999) Novel mechanisms for antisense-mediated regulation of gene expression.
Biochimica et Biophysica Acta. (1489): 3-18.
6. Barrett J.C, Miller P.S. and Ts’o POP (1974) Inhibitory effect of complex formation with
ologodeoxyribonucleotide ethyl phosphotriesters on transfer ribonucleic acid aminoacyiation. Biochemistry
(13): 4897-4906.
7. Bost F., McKay R., Dean N.M., Pota pova O. and Mercola D. (2000) Antisense methods for discrimina-
tion of phenotypic properties of closely related gene products: Jun kinase family. Methods Enzymol.
(314): 342-362.
8. Brummell D.A. and Harpster M.H. (2001) Cell wall metabolism in fruit softening and quality and its
manipulation in transgenic plants. Plant Mol. Biol. (47): 311-340.
9. Brummell D.A., Harpster M.H., Civello P.M., Palys J.M., Ben¬nett A.B. and Dunsmuir P. (1999)
Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabo-
lism during ripening. Plant Cell. (11): 2203-2216.
10. Caruthers M.H. (1985) Gene synthesis machines: D.N.A. chemistry and its uses. Science. 230 (4723):
281-285.
11. Clerq EDe, Eckstein F. and Merigan T.C. (1969) Interferon induction increased through chemical modifica-
tion of a synthetic polyribonucleotide. Science. (165): 1137-1139.
12. Crooke S.T. (1998) Basic Principles of Antisense Therapeutics, Antisense Research and Application,
Springer-Verlag, Berlin, Heidelber, New York, pp. 1-50.
13. Crooke S.T. (2000) Potential roles of antisense technology in cancer chemotherapy. Oncogens. (19): 6651-
6659.
14. Crooke S.T. (2004), Progress in antisense technology. Annual Review of Medicine. (55): 61-95.
15. Dandekari A.M., Teo G., Defilippi B.G., Uratsu S.L., Passey A.J., Kader A.A., Stow J.R., Colgan R.J. and
James D.J. (2004) Effect of down-regulation of ethylene biosynthesis on fruit flavor complex in apple
fruit. Transgenic Res. (13): 373-384.
16. Devor E.J. (2005) I.D. Tutorial: Antisense Technologies. Integrated D.N.A. Technologies.
17. Dias N. and Stein C.A. (2002) Antisense Oligonucleotides: Basic Concepts and Mechanisms. Molecular
Cancer. Therapeutics. (1): 347-355.
18. Fire A, S.Q. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello (1998) Potent and specific
genetic interference by double stranded R.N.A. in Caenorhabditis elegans. Nature. (391): 806-811.
 Antisense Technology 313

19. Gao W., Stein C.A., Cohen J.S., Dutschman G.E. and Cheng Y.C. (1989) Effect of phosphorothioate
homo-oligodeoxynucleotides on herpes simplex virus type 2-induced DNA polymerase. J. Biol. Chem.
(264): 11521-11526.
20. Gillespie D. and Spiegelman S. (1965) A quantitative assay for DNA-RNA hybrids with DNA immobi-
lized on a membrane. J. Mol. Boil. 12 (3): 829-842.
21. Goetz M.P., Knox S.K., Suman V.J., Rae J.M, Safgren S.L., Ames M.M., Visscher D.W., Reynolds C.,
Couch F.J., Lingle W.L., Weinshilboum R.M., Fritcher E.G., Nibbe A.M., Desta Z., Nguyen A., Flockhart
D.A., Perez E. and Ingle J.N. (2007) The impact of cytochrome P450 2D6 metabolism in women receiving
adjuvant tamoxifen. Breast. Cancer. Res. Treat. 101 (1): 113-21.
22. Goodchild J., Agrawal S., Civeira M.P., Sarin P.S., Sun D. and Zamecnik P.C. (1988) Inhibition of human
immunodeficiency virus replication by antisense Oligo-deoxynucleotides. Proc. Natl. Acad. Sci. USA (85):
5507-5511.
23. Gupta S. M., Srivastava S., Sane A.P. and Nath P. (2006) Differential expression of genes during banana
fruit development, ripening and 1-MCP treatment: presence of distinct fruit specific, ethylene induced and
repressed expression. Postharvest Biol. Technol. (42): 16-22.
24. Gupta S. M., Srivastava S. Gupta S., and Ahmed Z. (2008) Genetic manipulation of fruit ripening: using
antisense mRNA strategies. J. of Applied Biosciences. 34 (2): 115-123.
25. Gupta S., Kumar N., and Gupta S.M. (2009) Utility of chlorophyllose activity in mango (Mangijera
indica) peil for assessing state of ripening. Natl. Acad. Sci. Lett. 32 (182): 15-17.
26. Hamilton A.J., Lycett G.W. and Grierson D. (1990) Antisense gene that inhibits synthesis of the hormone
ehtylene in transgenic plants. Nature. (346): 284-287.
27. Haseloff J. and W.L. Gerlach (1988) Simple RNA enzymes with new and highly specific endoribonuclease
activities. Nature. (334): 585-591.
28. Heidenreich Olaf, Kang Shin-Heh, Xu Xiao and Nerenberg Michael (1995) Application of antisense
technology to therapeutics. Molecular Medicine Today. 1 (3): 128-133.
29. Herdewijn P. (2000) Heterocyclic modifications of oligonucleotides and antisense technology. Antisense
Nucleic Acids and Drug Development. (10): 117-121.
30. Ito M.K. (2007) ISIS 301012 gene therapy for hypercholesterolemia: sense, antisense, or nonsense? Ann
Pharmacother. 1 (10): 1669-78.
31. Jiménez-Bermúdez S., Redondo-Nevado J., Muñoz-Blanco J., Caballero J.L., López-Aranda J.M. and
Valpuesta V. (2002) Manipulation of strawberry fruit softening by antisense expression of a pectate lyase
gene, Plant Physiology. (128): 751-759.
32. Kemmer C., Neubauer P. (2006) Antisense RNA based down-regulation of RNaseE in E. coli. Microb Cell
Fact. (12): 5-38.
33. Knoester M., Linthorst H.J., Bol J.F. and van Loon L.C. (1997) Modulation of stress-inducible ethylene
biosynthesis by sense and antisense gene expression in tobacco. Plant Science. (126): 173-183.
34. Kurreck J. (2003) Antisense technologies: Improvement through novel chemical modifications. European
Journal of Biochemistry. (270): 1628-1644.
35. Marcus-Sekura C.J., Woerner A.M., Zon G. and Quinnan (1987) Comparative inhibition of chlorampheni-
col acetylttransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester,
methlyphosphonate and phosphorothioate linkage. Nucleic Acids Res. (15): 5749-5763.
36. Matsukura M., Shinozuka K., Zon G., Mitsuya H., Reitz M., Cohen J.S. and Broder S. (1987)
Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of
human immunodeficiency virus. Proceedings of the National Academy of Sciences USA. (84): 7706-7719.
37. Maxon J. and Weaver C. (2007) Antisense therapy: An overview. Current Topics In Oncology.
38. Miller P.S., L.T. Braiterman, and POP Ts’o (1977) Effects of a trinucleotide ethyl phosphotriester,
Gmp(Et)Gmp(Et)U, on mammalian cells in culture. Biochemistry. (16): 1988-1996.
314 Biotechnology in Medicine and Agriculture

39. Neckers L. and whitesell L. (1993) Antisense technology: biological utility and pratical consideration. Am.
J. Physiol. (265): 1-12.
40. Nielsen P.E. (2005) Systemic delivery: The last hurdle? Gene Therapy. (12): 956-957.
41. Oeller P.W., Lu M.W., Taylor L.P., Pike D.A. and Theologis A. (1991) Reversible inhibition of tomato
fruit senescence by antisense RNA. Science. (254): 437-439.
42. Redondo-Nevado J., Moyano E., Medina-Escobar N., Caballero J.L. and Muñoz-Blanco J. (2001) A fruit-
specific and developmentally regulated endopolygalacturonase gene from strawberry (Fragaria ´ anaassa
cv. Chandler), J. Exp. Bot. (52): 1941-1945.
43. Richard R. (2000) The application of antisense technology to medicine. The Ochsner Journal. (2): 233-
236.
44. Scherer L.J. and Rossi J.J. (2003) Approaches for the sequence specific knockdown of mRNA. Nat.
Biotech. (21): 1457-1463.
45. Sheehy R.E., Kramer M. and Hiatt W.R. (1988) Reduction of polygalacturonase activity in tomato fruit
by antisense RNA. Proc. Natl. Acad. Sci. USA. (85): 8805-8809.
46. Smith C.C., Aurelian I., Reddy M.P., Miller P.S. and Ts’o POP (1986) Antiviral effect of an oligo
(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type I
immediate early pre-mRNA 4 and 5. Proc. Natl. Acad. Sci. USA. (83): 2787-2791.
47. Smith C.J.S., Watson C.F., Ray J., Bird C.R., Morris R. Schuch W. and Grierson D. (1988) Antisense
RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature. (334): 724-726.
48. Smith C.J.S., Watson C.P., MOlTis P.C., Bird C.R., Seymour G.B., Gray J.E., Arnold C., Tucker G.A.,
Schuch W., Harding S. and Grierson D. (1990) Inheritance and effect on ripening of antisense polygalactu-
ronase genes in transgenic tomatoes. Plant Mol. BioI. (14): 369-379.
49. Souq F., Coutos-Thevenot P., Yean H., Delbard G., Maziere Y., Barbe J.P. and Boulay M. (1996) Genetic
transformation of roses, 2 examples: one on morphogenesis, the other on anthocyanin biosynthetic path-
way. ISHS Acta Horticulturae 424: II International Rose Symposium (eds. A. Morisot and P. Ricci) pp.
381-388.
50. Srivastava S. and Gupta S.M. (2008) Role of differentially expressed ripening related genes and promoters
in banana fruit: identified by mRNA DDRY – PCR. Res. Environ Life Sci. 1 (3): 81-90.
51. Tennant R.W., J.G. Farrelly, J.N. Ihle, B.C. Pal, F.T. Kenney, and A. Brown (1973) Effects of polyade-
nylic acids on functions of murine RNA tumor viruses. Journal of Virology. (12): 1216-25.
52. Thompson J.D. and Gillespie D. (1990) Current concepts in quantitative molecular hybridization. Clin
Biochem. 23 (4): 261-266.
53. Tracie L. Pierce, Anthony R. White, Geoffrey W. Tregear and Patrick M. Sexton (2005) Peptide-Oligo-
nucleotide hybrids in antisense therapy. Mini-Reviews in Medicinal Chemistry. (5): 41-55.
54. Ts’o P.O., Miller P.S. and Greene J.J. (1983) Nucleic acids alalogs with targeted delivery at the therapeutic
agents for development of target oriented anticancer drugs. Raven Press, New York. p. 189.
55. Van der Salm T.P.M., van der Toorn, C.J.G., Bouwer R., ten Cate C.H.H. and Dons H.J.M. (1997)
Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting
ability. Molecular Breeding. (3): 39-47.
56. Walder R.Y, and J.A. Walder (1988) Role of RNase H. in hybrid-arrested translation by antisense oligo-
nucleotides. Proceedings of the National Academy of Sciences USA. (85): 5011-5015.
57. Wan Q, Zhang X.G. and Song M. (2007) Fruit-specific RNAi-mediated Restraining Expression of Lcy
Gene to Enhance Lycopene Content in Tomatoes. Chinese Journal of Biotechnology. 23 (3): 29-434.
58. Wang D., Fan J. and Ranu R.S. (2004) Coning and expression of 1-aminocycloproane-1 carboxylate
synthase cDNA from rosa (Rosa x hybrida). Plant. Cell. Rep. (24): 422-429.
59. Wang T.W., Zhang C.G., Wu W., Nowack L.M., Madey E. and Thompson J.E. (2005) Antisense suppres-
sion of deoxyhypusine synthase in tomato delays fruit softening and alters growth and development. Plant
Physiol. (138): 1372-1382.
 Antisense Technology 315

60. Watson J. and Crick F. (1953) Molecular structure of nucleic acids; a structure for deoxyribose nucleic
acid. Nature. (171): 737.
61. Xiangqian L., Gasic K., Cammue B., Broekaert W. and Korban S.S. (2003) Transgenic rose lines harbouring
an antimicrobial protein gene, Ace-AMP1, demonstrate enhanced reistance to powdery mildew
(Sphaerotheca pannosa). Planta. (218): 226-232.
62. Yang S.F. and Hoffman N.E. (1984) Ethylene biosysnthesis and its regulation in higher plants. Ann. Rev.
Plant. Physiol. (35): 155-189.
63. Yoshihiko Tomita (2000) Application of antisense technology to urologic cancers. Molecular Urology.
4 (2): 55-59.
64. Zamecnik P.C. and M.L. Stephenson (1978) Inhibition of Rous sarcoma virus replication and cell transfor-
mation by a specific oligodeoxynucleotide. Proceedings of the National Academy of Sciences USA. (75):
280-284.
65. Zhang W., Zhang H. and Xing L. (2006) Antisense oligonucleotide of hypoxia-inducible factor-1alpha
suppresses growth and tumorigenicity of lung cancer cells A549. J. Huazhong Univ. Sci. Technolog Med.
Sci. 26 (4): 448-450.
66. Zuo S., Luo J., Liu M., Xu L., Dong J., Guo W. and Zou S. (2008) Suppressing effects of down-regulating
DNMT1 and DNMT3b expression on the growth of human cholangiocarcinoma cell line. J. Huazhong
Univ. Sci. Technolog. Med. Sci. 28 (3): 276-80.

View publication stats