1. Antisense technolgy:
Part 2: continue

2. The Cycle of Oligonucleotide - Directed RNase H Cleavage
A single oligonucleotide can direct RNase H to cleave and thereby degrade many target mRNA molecules. If RNase H cleaves a mRNA molecule it cannot be translated into protein because the message has become degraded. Thus oligonucleotides, which efficiently direct RNase H to cleave their target mRNA may be able to inhibit production of the protein coded by that mRNA.  The figure below shows that under conditions where oligonucleotide, mRNA and RNase H may move freely in solution:
1-Target mRNA (yellow) hybridizes to the antisense oligonucleotide (red).
2.  2- RNase H complexes with the hybrid and a reaction ensue in which only the mRNA strand is cleaved.
3.  3- The enzyme - product complex dissociates releasing degraded mRNA (and potentially active RNase H).
4.    4- Free, intact, antisense oligonucleotide is available to hybridize to another mRNA molecule, and start the cycle again.

4. Structures and Summary of Properties of antisense oligos.
 Phosphodiester, Phosphorothioate, Methylphosphonate
 Phosphorothioate and methylphosphonate analogues arise from modification of the phosphate groups in the oligonucleotide backbone. In the figure below we can see that the phosphodiester phosphate O- is replaced by a sulphur (S-) group in phosphorothioate structure and a methyl (CH3) group in methylphosphonates.

5. Phosphodiester Phosphorothioate Methylphosphonate
Both phosphorothioate and methylphosphonate oligonucleotides are much more resistant to degradation by nucleases than natural phosphodiester structures. However, both have undesirable characteristics. Phosphorothioate oligonucleotides have been widely reported to interact with cellular proteins and thereby induce a variety on non - antisense biological effects. On the other hand, methylphosphonate compounds do not induce such non - specific effects, but hybridize to complementary RNA rather weakly and are incapable of directing the activity of RNase H.

6. 2- Chimeric Oligonucleotides chimeric' oligonucleotides are constructed from more than one sort of structural element. Chimeric methylphosphonate / phosphodiester oligonucleotides are resistant to exonucleases, those which degrade the molecule from an end and which constitute the majority of the nuclease activity in biological fluids and are capable of directing the activity of RNase H when they are delivered by streptolysin O into living cells. The general structure of chimeric methylphosphonate / phosphodiester oligonucleotides is displayed in the figure below.
Methylphosphonate------Phosphodiester----- Methylphosphonate

7. 3- Morpholino subunits

8. A dominant consideration in the design of most antisense oligos has been to devise a structure that provides resistance to nucleases while still resembling natural nucleic acids as closely as possible. Although oligomers assembled from such Morpholino subunits differ substantially from DNA, RNA, and analogs thereof, initial modeling studies carried out in 1985 suggested that such novel Morpholino-based oligomers might constitute useful and highly cost-effective antisense agents. The simple and inexpensive ribose to morpholine conversion replaces two poor nucleophiles (the 2' and 3' hydroxyls) with a single good nucleophile (the morpholine nitrogen) and allows oligo assembly via simple and efficient coupling to the morpholine nitrogen without the expensive catalysts and postcoupling oxidation steps required in the production of most DNA-like antisense oligos. It is noteworthy that in spite of the relatively low nucleophilicity of the morpholine nitrogen (pKa = 5.75), we still typically achieve coupling efficiencies of 99.7% without using catalysts.

9. Inserting Antisense into Cells:
Endocytosis: 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.
Microinjection: the antisense molecule would be injected into the cell. The yield of this method is very high, but because of the precision need to inject a very small cell with smaller molecules only about 100 cells can be injected per day. Liposome Encapsulation: This is the most effective method, but also a very expensive one. Using products such as LipofectACE(TM) to create a cationic phospholipid bilayer that will surround the nucleotide sequence can achieve liposome encapsulation. The resulting liposome can merge with the cell membrane allowing the antisense to enter 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.

10. Advantages of an Antisense Approach
The main advantages that an antisense approach to therapy offers are specificity and point of attack. Antisense targets can be selected that are unique to the gene whose expression is to be controlled. Hence only that gene's expression is inhibited. This is especially important for diseases like viral infections and cancers, which employ normal cell functions in the disease process. It has been difficult to devise therapeutic strategies against these diseases without also disabling normal cell functioning. Antisense nucleic acids targeted toward a gene that is diverting these normal cell functions, however, can specifically impair the Disease State without affecting cell function. An antisense approach, therefore, has fewer side effects and offers real therapeutic promise for certain cancers and viral diseases.
     

11. Another advantage to an antisense approach is that it interferes at the source of the disease. That is, it interferes with the formation of unwanted proteins, rather than stopping these proteins from functioning. In addition, technically it is easier to define nucleic acid targets than it is to define protein targets. Information about gene structure is being amassed at an enormous rate, whereas information about protein structure is much harder to obtain. To illustrate, the genetic sequence of the human genome is expecting to be completely known in fifteen years (HGP). It will be considerably longer, however, before it is understood what all it encodes, and longer still before the spatial structure of very many of the encoded proteins is known.

12. Application of Antisense
1- Commercial Opportunities of Antisense
Because an antisense approach is generally applicable to the regulation of any gene, it has broad commercial potential and can address markets of considerable magnitude. In addition to its usefulness in therapeutics, it can have a strong impact on agriculture and the bioprocess industry.
2- Therapeutics: The extreme specificity of antisense offers real therapeutic promise for cancer and viral infections, vaccines, thereby opening up new markets of considerable size. By the end of the century it is estimated that greater than one million new cases of cancer will likely be diagnosed in the U.S. and more than a half million people will die of this disease. Last year greater than $2 billion was spent on cancer chemotherapeutic products and this market is growing around 25% per year.

13.      Anti-viral drugs and vaccines are estimated to be about a third of the approximately $7 billion spent worldwide on anti-infective last year, indicating both the difficulty in finding effective anti-viral therapy and their market potential. For AIDS alone the statistics are staggering. The Centers for Disease Control estimate that in the United States 1 to 1.5 million people (1 in 250 persons) are infected with HIV. Current estimates indicate that, worldwide, as many as 8 to 10 million persons may carry this virus. According to Public Health Service statistics, the cumulative number of reported AIDS cases is currently greater than 500,000 and half of these have been reported since 1993.

14. 3- Agriculture: The use of antisense technologies to develop agricultural animals or crops that are resistant to viruses or other diseases also has considerable commercial significance. For example, chickens resistant to viral diseases such as Marek's Disease and Newcastle Disease Virus could strongly impact poultry production. It has been estimated that the annual cost of Marek's Disease is $940 million due to vaccination costs and lost meat and egg production on the worldwide. Plant viral diseases are believed to reduce yields 10-20% across all plant crops. In the U.S. it is estimated that viruses are responsible for a loss of $1.5 to $2 billion in crop value each year, primarily in grains. Antisense could also be used to develop plants resistant to those fungi that require the expression of certain plant genes before they can successfully invade that plant. This could reduce the $350 million spent annually in the U.S. on fungicides.

15.      Applications of this technology, which alter the nutritional content, or shelf-life of foods would also have significant commercial impact. One possibility is reduced-cholesterol eggs.
In plants antisense genetic technology can also be used to control genes that promote ripening and softening of fruits and vegetables, thereby providing considerable savings in picking, transport and storage as well as potentially expanding the seasonal availability of some fruits and vegetables. Similarly, antisense genetic technology could conceivably be used to improve the ratio of monosaturated fats to polyunsaturated fats in plant oils.

16. 4- Bioprocessing Industry:
With the advent of genetic engineering the number of commercially important materials being produced from fermentation and cell culture is increasing rapidly. In the pharmaceutical area alone, it is anticipated that U.S. revenues from products obtained by biological processes will total $12 billion by the year The yields of desired products can be improved with an antisense genetic strategy that inhibits interfering cellular reactions. This approach has already doubled the yield of Factor VIII from genetically engineered mammalian cells. The annual worldwide market for Factor VIII is estimated at $250 million.
     On worldwide basis products attainable from plant cell culture for use in medicinals, flavors, fragrances and agricultural chemicals comprise about a $750 million market. Important medicinal products in this category include corticosteroids and codeine. It is likely that the yield of these products can be improved by culturing cells in which antisense genes reduce competing pathways.

17. 5- Antisense for functional genomics and drug target
validation
One application for antisense technology is to validate drug targets. Although the phenotypes of many diseases are well known, identification of the genes responsible for those phenotypes remains a major hurdle in the drug development process. Historically, drug targets were identified and validated by screening large numbers of small-molecules designed to inhibit the function of particular genes. The development of small molecule inhibitors requires substantially more information than is required to design antisense oligo -nucleotides. Many small-molecules interact with multiple members of a gene family, confounding the validity of the intended gene as a drug target. Antisense technology enables researchers to elucidate the roles of gene products within cellular pathways by specific inhibition of individual or simultaneous inhibition of multiple members of a gene family in their experimental system.

18. During the past few years, antisense technology has played a key role in drug discovery and target validation efforts based on sequence information gathered from the HGP .Many pharmaceutical companies are employing the technology to identify the function of novel sequences mined from the private and public databases. The versatility of the antisense approach makes it attractive for use in drug discovery efforts. Antisense oligonucleotides, in conjunction with the appropriate phenotypic assay, provide a rapid method for screening potential drug targets for various diseases (next Fig.).

19. - Alzheimer’s disease: In 1999 antisense technology was used to implicate ß-secretase as a novel and potentially important drug target for the treatment of Alzheimer’s disease. In carefully controlled experiments, it was shown by both groups that inhibition of the ß-secretase target by several antisense oligonucleotides that targeted different sites within the ß-secretase mRNA, resulted in a decrease in amyloid-ß peptide processing. No effects were observed with the use of reverse sequence or mismatch control oligonucleotides. Recently, it was demonstrated that mice deficient in the Alzheimer’s ß-secretase (BACE) have normal phenotype and abolished ß-amyloid generation, confirming BACE as a valid drug target.

20. 7- Determination of gene function in vivo
Once a potential drug target has been identified in a cell culture screen, the next step is to confirm the validity of the target through in vivo evaluation. The gold standard for determining gene function in vivo has been the use of genetic-knockout technology. Although genetic knockouts can be used to define clearly the role of a gene within the whole animal, the technology is not without limitations. The major limitations of knockout technologies for in vivo target validation are the length of time required to create and characterize the mutant strain, and the possibility of generating a lethal embryonic mutation, which precludes the evaluation of certain genes.

21.  Antisense oligonucleotides are an alternative method for identification of gene function in vivo. The effects of inhibition of target genes on the function of the whole animal can be determined rapidly. Also, the function of genes at various points in development, ranging from embryonic stages to adult animals, can be evaluated using antisense technologies. In the past few years, antisense technologies have been used successfully for the validation of multiple targets in in vivo models. For example, interleukin(IL)-5 is a cytokine that has been implicated in the migration of eosinophils in the asthmatic lung. Using antisense oligonucleotides that target IL-5 in a well-characterized murine model of asthma, it was demonstrated that a reduction in IL-5 protein corresponded with decreased eosinophilia and improved airway function.

22. The proto-oncogene c-Myc is known to play a crucial role in the regulation of cell proliferation, differentiation and apoptosis. However, the role of c-Myc in the process of tissue regeneration is not clearly understood. Using a liver regeneration model after partial hepatectomy, it was showed that inhibition of Myc by antisense oligomers caused a dose-dependent and sequence-specific reduction of liver regeneration. In another study focusing on the effects in the liver, it was shown that antisense oligonucleotides targeting Fas offered a protective effect in a murine model of hepatitis. It should not be overlooked that antisense oligo -nucleotides used for in vivo drug target validation could be further developed as therapeutic agents. Several antisense compounds are currently in clinical trials for the treatment of a broad spectrum of diseases, ranging from viral infection to cancer10 (Table 1). Whereas antisense-based therapeutics might be practical for target genes expressed in tissues in which oligonucleotides accumulate at high levels (e.g. liver), efficient delivery of the oligonucleotides to other target tissues could limit the therapeutic potential of antisense technologies.

24. "In anaplastic astrocytoma, AP as a monotherapy is actually clearly superior to temozolomide," Prof. Albert Wong, M.D., Stanford University, California, U.S.A. commented on the international Phase IIb study with the TGF-beta 2-inhibitor AP 12009, under development by Antisense Pharma. (forcentral nervous system tumors )

25. Multiple Second-Generation Antisense Drugs Show Potential as New Treatments for Diabetes and Metabolic Disease
Isis Pharmaceuticals
Isis Pharmaceuticals, Inc. (Nasdaq: ISIS - News) announced results from fourteen preclinical studies demonstrating potent, selective antisense inhibitors of gene targets, directly associated with therapeutic potential in a variety of preclinical models of metabolic diseases, including diabetes, non-alcoholic fatty liver disease (NASH) and metabolic syndrome. Findings from the studies were presented by Isis and several collaborators this week during the American Diabetes Association's (ADA) 65th Scientific Sessions in San Diego.

26. Example antisense therapies
Cytomegalovirus retinitis
Fomivirsen (marketed as Vitravene), was approved by the U.S. FDA in Aug 1998 as a treatment for cytomegalovirus retinitis.
Hemorrhagic fever viruses
In early 2006, scientists studying the Ebola hemorrhagic fever virus at USAMRIID announced a 75% recovery rate after infecting four rhesus monkeys and then treating them with an antisense Morpholino drug developed by AVI BioPharma, a U.S. biotechnology firm. The usual mortality rate for monkeys infected with Ebola virus is 100%.

27. In late 2008, AVI BioPharma successfully filed Investigational New Drug (IND) applications with the FDA for its two lead products for Marburg and Ebola viruses. These drugs, AVI-6002 [3] and AVI-6003 are novel analogs based on AVI's PMO antisense chemistry in which anti-viral potency is enhanced by the addition of positively-charged components to the morpholino oligomer chain. Preclinical results of AVI-6002 and AVI-6003 demonstrated reproducible and high rates of survival in non-human primates challenged with a lethal infection of the Ebola and Marburg viruses, respectively.[4]

28. Cancer
Also in 2006, German physicians reported on a dose-escalation study for the compound AP (a phosphorothioate antisense oligodeoxynucleotide specific for the mRNA of human transforming growth factor TGF-beta2) in patients with high grade gliomas. At the time of the report, the median overall survival had not been obtained and the authors hinted at a potential cure.
HIV/AIDS
Starting in 2004, researchers in the US have been conducting research on using antisense technology to combat HIV.
In February 2010 researchers reported success in reducing HIV viral load using patient T-cells which had been harvested, modified with an RNA antisense strand to the HIV viral envelope protein, and re-infused into the patient during a planned lapse in retroviral drug therapy.
High cholesterol
In 2010 mipomersen successfully completed phase 3 trials for some types of high cholesterol

29. Conclusion
Antisense technology provides a rapid and specific method for determination of gene function, both in vitro and in vivo.
The antisense approach could reduce the cost of drug discovery by expediting the identification of lead targets for pharmaceutical intervention.
Many of the limitations of first-generation antisense oligomers33 have been overcome through advances in the technology leading to improved specificity and enhanced the stability of oligo nucleotides.
Transfection techniques have also been improved that enable delivery of antisense oligonucleotides to cells more reliably and with less toxicity than was previously possible.
Antisense technology is now proven as a viable option for systematic and high-throughput determination of gene function and drug target validation.