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Submitted to: Mr. Harsh Submitted by: BHAWANA KUMARI Btech.(biotech) Roll.no. 50
I would like to express my gratitude to all those who gave me the possibility to complete this term work. I want to thank the department of BIOTECHNOLOGY my friend’s kiran and sakshi for giving me permission to commence this work in the first instance and to use the research data.
CONTENTS: 1. INTRODUCTION 2. HOW IT WORKS 3. DEVELOPMENTS (a)EFFICIENCY 4. STRATEGY AND APPLICATIONS 5. OBSTACLES OF ANTISENSE TECHNOLOGY 6. BIBLOGRAPHY
In antisense technology, synthetically-produced complementary molecules seek out and bind to messenger RNA (mRNA) 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. 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 resulting from the binding of the antisense oligonucleotide to a unique gene sequence. 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.
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. Initially, cellular nucleases dramatically reduce the effectiveness of antisense oligonucleotides by rapidly degrading these molecules after administration. These obstacles can be overcome in applications utilizing synthetic oligonucleotides by altering the nature of the phosphodiester bond by replacing an 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 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 counterparts. 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 to cancer gene therapy
How does Antisense Technology Work
The therapeutic objective of antisense technology is to block the production of disease-causing proteins. This is achieved by creating a synthetic "antisense" or complementary nucleotide sequence of DNA or RNA that interacts with, and binds to the "sense" or original mRNA sequence. This "mRNA - antisense complex" can no longer be translated and the disease-causing protein cannot be produced. .Biotechnology
and Antisense Technology
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 diseasecausing proteins. However, antisense drug therapies inhibit the production of the disease-causing proteins altogether.
Antisense Therapies in Development
Applications of antisense technology in therapeutics are expected to increase substantially as the link between diseases and genes are made. 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. Antisense is an important technology for drug discovery and development. 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. Isis scientists have worked systematically and rigorously to address the questions associated with the development of this new drug discovery platform. How can specificity and potency of antisense drugs be increased? 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:
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. One of the most alluring attributes of antisense is its specificity. Antisense drugs interact with their intended target based on information in the genetic code. 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. Based on their specificity, antisense drugs have the potential to be safer than other types of drugs.
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. Isis scientists have performed hundreds of experiments to determine how and where antisense drugs distribute throughout the body. Research shows antisense drugs accumulate in specific organs and tissues, so Isis’ drug discovery research is focused on diseases that are associated with targets expressed in the liver, kidney, spleen, bone marrow and fat cells. Isis’ drug development programs are aimed at treating
cardiovascular, metabolic and inflammatory diseases. Isis’ partners are focused in disease areas such as ocular, viral and neurodegenerative diseases, and cancer. The Company’s researchers have also made significant progress in developing new formulations (aerosol, enema, and intrathecal, intravenous, subcutaneous, intravitreal, oral and topical) of antisense drugs that expand the potential for antisense.
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 medicinal chemistry is constant, with only the order of the drug’s nucleotides changing to make the drug specific for the target of interest. 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. Efficiency: The benefit of a more rational approach to drug discovery is efficiency. The efficiency 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. The efficiency of antisense technology is also realized in the area of manufacturing. Advances in process chemistry over the course of the Company’s history has resulted in a dramatic reduction in the cost of Isis’ drugs. Isis built a state-of-the-art, commercial scale manufacturing facility, which was completed in February 2003. The facility is capable of producing hundreds of kilograms per year of antisense drugs.
Antisense Drug Technology Principles: Strategies, and Applications :
This state-of-the-art reference provides comprehensive coverage of the development of antisense oligonucleotides to inhibit cancer cells as well as those involved in
infectious, inflammatory, and immune-mediated diseases-highlighting new tools and technologies in medicinal chemistry, RNA biochemistry, and molecular and cellular biology to produce new therapeutic compounds. Presents previously unpublished data on the use of antisense technology to dissect pharmacological processes and confirm the roles of various genes. Showcasing the benefits of antisense drug use, including reduced toxicity and earlier disease detection, Antisense Drug Technology discusses
• • • • • •
novel formulations of antisense drugs practical methods to design effective isotype selective inhibitors molecular mechanisms of antisense drugs mRNA as a current biological template modern post receptor binding mechanisms and more! With contributions by over 60 seasoned experts in the field and containing more than 3000 helpful references, tables, drawings, and photographs, Antisense Drug Technology is an illuminating source for organic, medicinal, and pharmaceutical chemists; biochemists; geneticists; hematologists; oncologists; molecular and cell biologists; virologists; immunologists; and medical school and graduate students in these disciplines.
Antisense, Heat Shock Proteins and the Heart:
Antisense technology provides a tool with which to dissect the components of the stress response. There are two known endogenous sets of protective proteins, the heat shock proteins (Hsps) and the antioxidants, such as superoxide dismutase;1–4 components of both sets of proteins have been found to be induced in different stress settings, such as heat shock and ischemia. At the current time our ability to manipulate expression of these important genes is limited. The known stimuli induce multiple different stress response genes, which curtails our ability to understand the function of each of these genes. One approach to this question has been overexpression of individual Hsp, such as Hsp72 and Hsp27, each of which has now been shown to protect against injury such as ischemia, hypoxia, or heat.5–10 However, overexpression prior to injury does not clarify the role of a given protein in the endogenous stress response. Understanding this role increases our comprehension of protein function and also provides insight into the mechanisms of injury. Antisense technology permits selective inhibition of single genes. Such an approach allows the analysis of the contribution of individual heat shock proteins and antioxidant genes to cardiac protection.
Applications of Antisense:
Applications of antisense technology are very diverse. It was first successfully used in plants. One example of antisense technology that is currently available is the Flavr Savr tomato. Antisense was used to block the enzyme that is involved in
spoilage, thereby increasing the length of time a tomato could be sold. Antisense technology is now used in mammalian cells. Promising fields of study for antisense technology in humans include cancer gene therapy and AIDS. In cancer treatment, antisense is constructed in a way that it will bind with the mRNA from the PKC alpha gene (Conrad 1997). This gene is targeted because PKC alpha kinase is more sensitive in cancer cells. The treatment has resulted in a 50% decrease in the size of the tumor from ovarian cancer in one patient and stabilization of the tumor's growth in other patients (Conrad 1997). Tests are still being administered to determine the possibility of future use of the antisense treatment on a wider scale since the previous trial has shown antisense to be well- tolerated (Conrad 1997)
. Application of Antisense Technology
In a seminal biological insight 50 years ago, James Watson and Francis Crick proposed a structure for the genetic material deoxyribonucleic acid, or DNA. They proposed that DNA consists of two deoxyribonucleotide molecules each having a 5´end and a 3´-end, which define a polarity for each deoxyribonucleotide strand. The two strands, when bound together in antiparallel orientation (that is, the 3´-end of one being juxtaposed to the 5´-end of the other) compose a complete DNA molecule. The two strands are bound together by pairing among the four complementary bases in each strand such that adenine in one strand binds to thymine in the second, and cytosine in one binds to guanine in the second. Thus, all the genetic information is contained in each strand, written in what might be called a mirror image in each strand. This structure permits the transmission of genetic information by allowing a complementary strand to be produced for either single strand. One half (one strand) can therefore produce an entire DNA molecule, as occurs during cell divisio.Because each triplet set of nucleotides on a strand of DNA encodes an amino acid, this structure is also the basis of protein synthesis. In this process, one or another portion of one strand (a gene) is copied by ribonucleic acid polymerase II producing a complementary molecule of ribonucleic acid, or RNA. This messenger RNA (mRNA), therefore, contains essentially the same information that is contained in the gene that has been transcribed. The RNA molecule is processed in the nucleus such that certain sequences (introns) are excised leaving a smaller, mature RNA molecule. After intron excision and additional processing, the RNA molecule is exported to the cell cytoplasm where it directs protein synthesis at the ribosome. Because RNA is relatively unstable in the cell, the amount of protein synthesized is dependent on not only the rate and duration of RNA production from the gene but also on factors that stabilize or lengthen the lifespan of the RNA in the cytoplasm.
Therapeutic Application of Antisense Technology
A second application of this technology, and one that is potentially of more immediate relevance to the practicing physician, is the use of this technology in therapy). In principle, antisense oligonucleotides complementary to viral RNAs can suppress a wide variety of viral infections; a tremendous amount of research is
ongoing in this area. Similarly, antisense oligonucleotides directed towards the products of oncogenes can play a role in reducing the growth of cancer cells, and this lead is being hotly pursued. Perhaps the most widely discussed application of antisense technology lies in its applications to gene therapy. In this case, a variety of vectors is used to introduce antisense-encoding genes into a large number of cells in a patient or animal to produce long-term inhibition of a protein. For example, in animal models the introduction of vectors encoding antisense angiotensin II receptor sequences results in long-term normotension in spontaneously hypertensive animals. These are but a few of the possible applications of antisense technology. As familiarity with the relevant chemistry increases, it is likely that more effective oligonucleotides and gene vectors will be developed, thereby providing the ability to interfere at will with the translation of specific mRNAs.
Triplex Antisense Technology
In the face of all this progress, still newer technologies are being developed based on concepts related to antisense biology. For example, it is known that oligonucleotides can, in certain instances, bind to duplex DNA molecules through an unusual kind of base pairing. In this triplex binding mode, oligonucleotides insert themselves into the major groove of the DNA double helix on a reasonably specific basis determined by the nucleotide sequence of the target DNA. This triplex technology provides the opportunity to reduce gene transcription itself rather than to destroy mRNA once it is produced. Because the triplex oligonucleotides can be made to permanently alter the DNA after localizing to specific target sites, the technology actually has the potential to permanently silence genes. In our laboratory, we have used this technology in an effort to produce triplex binding to one of the consensus binding sites of the antitumor protein p53. The protein p53 is in normal cells and is turned on when cells become damaged or malignant, impairing their growth and preventing the reproduction of faulty cells. Certain cancer cells lack p53 function, which allows continued growth and reproduction. The p53 protein binds to specific DNA sequences, and we speculated that triplex oligonucleotides designed to bind to those sequences might mimic p53 effects. We subsequently demonstrated that the use of triplex oligonucleotides will suppress cell growth in certain cancer cells lacking normal p53 function, thus raising the possibility that triplex biology may lead to the development of effective anticancer agents.
It has recently been shown that double-stranded RNA in the cytoplasm triggers an as yet poorly understood cascade of events leading to the suppression of the transcription of the gene producing the specific mRNA involved in the cytoplasmic
RNA duplex. This could potentially lead to the development of new pharmacological agents. Antisense technology is a formidable tool for investigating physiologic and pathologic processes. In addition, it is soon likely to become a mainstay of therapy, particularly in infectious diseases, with wider applications in the future as gene therapy techniques are developed further. Antisense pharmaceuticals will soon be available for the routine care of patients and are expected to prove to be effective, specific agents with favorable therapeutic profiles.
Mechanisms & Applications Of Gene Silencing:
The down regulation and suppression of genes by both antisense and sense constructs are powerful and widely used techniques in molecular biology. They have been employed to investigate the metabolic role of enzymes, to identify functions for unknown genes and to investigate methods for treating diseases. In plants, transgenic sense and antisense crops are now being grown commercially. The advent of recombinant DNA technology has heralded rapid progress in our understanding and manipulation of gene control. This has facilitated the exploitation of powerful genetic engineering techniques in the fields of agriculture, food science and medicine. One very important aspect of this gene manipulation is the ability to selectively switch genes off. This gene silencing can be brought about by either of two methods: antisense RNA or sense co-suppression. The first products of gene silencing technology in eukaryotes, namely genetically manipulated tomatoes, have recently entered the marketplace and there can be little doubt that the application of this technology will be greatly extended in the future. This conference brought together experts from a wide range of disciplines to discuss the mechanisms and applications of gene silencing. These included molecular biologists, microbiologists, biochemists and physiologists with interests in the plant, animal and medical sciences. The conference addressed the perplexing question of exactly how gene silencing occurs at the molecular level. Current theories were explored and a diverse range of experimental models to address this question were presented. Current and future applications of this technology in plant, animal and medical science were discussed. In particular the applications of gene silencing to induce viral resistance in plants and to treat cancers in animals were examined.
A medical application of antisense oligomers against cancers and viral infections:
Gene-specific nucleic acid therapy has gone from theory to practical possibility in a very short time. The new DNA and RNA agents are intended to stop the growth of cancerous cells or the production of HIV from infected cells. In the present review,
the clinical and biomedical applications that appear to be plausible at that time, are discussed for developing therapeutic agents in a next decade by presenting a few examples of the wide variety of current studies in this field. I believe that this approach provides a powerful tool for elucidating the role of a particular gene and for therapeutic To use antisense nucleic acids as pharmaceutical reagents, clear explanations at the molecular level must be provided as to how they work to achieve specific effects. Unfortunately, there is a great deal of uncertainty. We report here the basic concepts and problems of antisense technology and the molecular understanding of it to develop the pharmaceutical use against cancers and viral infections. A gene analysis using antisense oligomers always involves the following problems: (1) base pair specificity; (2) stereoisomer specificity; (3) stability and nuclease resistance of the sense- antisense duplexes; (4) cell membrane permeability and targeting; (5) safety; (6) preparation of large amounts of oligomers. APPLICATION TO THERAPEUTICS: Antisense oligonucleotides inhibit gene expression by binding in a sequence-specific manner to an RNA target. Modern nucleotide chemistry has enabled the synthesis of chemically modified oligonucleotides that are highly resistant to nuclease degradation. Among other applications, these agents are currently being evaluated as potential antiviral and anticancer drugs. However, several unsolved problems remain. Poor efficiency of delivery to cells, tissue toxicity and antisense-independent biological effects of oligonucleotides currently limit the widespread application of oligonucleotides to human disease. This article reviews some of the applications of antisense oligonucleotides and discusses problems associated with these applications.
Application of Antisense Technology in vitro
Antisense technology has been applied successfully in two general areas. The first is in fundamental research where the introduction of antisense oligonucleotides can help determine the role of a specific gene in a specific physiological process). For example, our laboratory has been interested for some time in the idea that local components of the renin angiotensin system can be produced by specific cells. We hypothesized that the production of angiotensin II by cells feeds back on those cells resulting in cell growth and other changes. In our view, this tissue (cellular renin angiotensin system) could therefore potentially play a role in a wide variety of cardiovascular disorders, including atherosclerosis and vascular hypertrophy. It is difficult to demonstrate that a cellular system is operative in any given process, however, because a circulatory renin angiotensin
system exists that produces angiotensin II in tissue culture medium as well as in tissues. To approach this problem we developed oligonucleotides to inhibit the synthesis of angiotensinogen, the substrate from which cells make angiotensin II. We were able to demonstrate that the application of these oligonucleotides, but not of scrambled oligonucleotides (control oligonucleotides of identical composition but scrambled sequence), resulted in a decline in cell growth. The introduction of angiotensin II to the cells restored this growth. Thus, through the application of antisense technology, we were able to demonstrate the biological principle that cells can make their own angiotensin II with growth promoting effects. We extended this antisense work to certain cancers and demonstrated, for the first time, that neuroblastoma cells, for example, also possess similar cellular renin angiotensin systems that regulate their growth These studies, and many like them, represent the application of antisense technology to research.
The Wonderful World of Antisense Technology From DNA to Protein Expression: Protein molecules are the expressions
of a gene. However, to get to a protein the cell must undergo two complex processes, transcription and translation. Transcription is the process in which an RNA copy is made of the DNA. In order to get the copy, many enzymes such as polymerase, helicase, exonuclease, ligase and single stranded binding proteins work together to unwind the double helix and match the base pairs of RNA (adenine, guanine, cytosine and uracil) to the DNA. Once the copy is made, the RNA molecule, which is now in the heterogeneous nuclear RNA (hnRNA) mode, is still not ready to go express the gene by making a protein. The hnRNA must be spliced to remove non coding sequences, and protected from the cellular environment with a 5' cap and poly- A tail. Finally, the hnRNA is transported out of the nuclear membrane and into the cytoplasm where it achieves the status of mRNA. In the cytoplasm, the mRNA molecule hooks up with ribosomes where the protein production can start. Every three nucleotides in the mRNA molecule codes for a specific amino acid and are appropriately called a codon. The codon pairs with an anti codon of tRNA that has attached to an amino acid. In this manner a polypeptide chain is formed. It will eventually twist and contort itself into a unique configuration which aids in the function of the protein. Occasionally, a bad mRNA molecule is synthesized so that the resulting protein can not function properly. Abnormalities of proteins cause many diseases that afflict humans. Therefore, it seems logical to conclude that if the expression of these malfunctional proteins could be stopped, the sources of disease would be obliterated and the disease will be treated, if not cured. This idea is the basis for antisense technology. The Basics of Antisense: A sense strand is a 5' to 3' mRNA molecule or DNA molecule. The complementary strand, or mirror strand, to the sense is called an antisense (What is Antisense 1998). Antisense technology is the process in which the antisense strand hydrogen bonds with the targeted sense strand. When an antisense strand binds to a mRNA sense strand, a cell will recognize the double helix as foreign to the cell and proceed to degrade the faulty mRNA molecule, thus preventing the
production of undesired protein. Although DNA is already a double stranded molecule, antisense technology can be applied to it, building a triplex formation (Fritz 1996). A DNA antisense molecule must be approximately seventeen bases in order to function, and approximately thirteen bases for an RNA molecule. RNA antisense strands can be either catalytic, or non catalytic. The catalytic antisense strands, also called ribozymes, which will cleave the RNA molecule at specific sequences. A non catalytic RNA antisense strand blocks further RNA processing, i.e. modifying the mRNA strand or transcription (Fritz 1996). The exact mechanism of an antisense strand has not been determined. The current hypotheses include blocking RNA splicing, accelerating degradation of the RNA molecule, preventing introns from being spliced out of the hnRNA, impeding the exportation of mRNA into the cytoplasm, hindering translation, and the triplex formation in DNA (Fritz 1996). The two figures below illustrate the fact that no consenus has been reached concerning how antisense accomplishes the reduction of protein synthesis.
Inserting Antisense into Cells: Endocytosis- One of the simplest methods to get nucleotides in the cell, it relies
on the cell's natural process of receptor mediated endocytosis. The drawbacks to this method are the long amount of time for any accumulation to occur, the unreliable results, and the inefficiency (Dantus 1997).
Micro- Injection- As the name implies, the antisense molecule would be injected
into the cell. The yield of this method is very high, but because of the precision needed to inject a very small cell with smaller molecules only about 100 cells can be injected per day (Dantus 1997).
Liposome Encapsulation- This is the most effective method, but also a very
expensive one (Dantus 1997). Liposome encapsulation can be achieved by using products such as LipofectACE(TM) to create a cationic phospholipid bilayer that will surround the nucleotide sequence. The resulting liposome can merge with the cell membrane allowing the antisense to enter the cell.
Electroporation- The conventional method of adding a nucleotide sequence to a
cell can also be used. The antisense molecule should traverse the cell membrane after a shock is applied to the cells.
Obstacles of Antisense:
The successful result of any of these methods is reduced protein production as long as the surrounding conditions are favorable (Driver). However, there are more
problems that need to be overcome then just inserting the antisense molecule into the cell. If antisense is being used as a treatment in the human body, it might be degraded before stopping any protein production if it is unmethylated because the body would recognize it as an invader (Dantus 1997). When antisense is used in living organisms, there are also many complications that can arise such as high blood pressure and a low white blood cell count (Dantus 1997).
http://www.isispharm.com/antisense_tech.html http://www.bio.davidson.edu/Courses/Molbio/MolStudents/01suschultz/homepage.ht ml http://www.elsevier.com/wps/find/bookdescription.cws_home/675274/description#de scription http://books.google.co.in/books?hl=en&id=96jrNnqPRkC&dq=antisense+technology&printsec=frontcover&source=web&ots=86ddMb1Zp O&sig=AHWGbZ9wPK_JT2Yztt3ay3pGAYw&sa=X&oi=book_result&resnum=9& ct=result
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