In living organisms, DNA carries all of the instructions for building the proteins that make up an organism, coded in small sections called genes. DNA consists of a constant backbone made of sugar and phosphate molecules as well as variable nucleotides or bases (Adenine, Thymine, Guanine, Cytosine, commonly known as A, T, G and C). DNA resembles a ladder, forming a twisted, double-stranded structure with the nucleotides making up the rungs of the ladder and the backbone forming the sides of the ladder. One strand of the ladder (sense) matches exactly with its complementary strand (antisense); the nucleotides that make up the rungs of the ladder are very specific in how they match each other. A always partners with T and C always partners with G.
The sequence or order of these nucleotides establishes the cell’s recipe for making proteins. In a process called transcription, DNA is used as a template to manufacture an RNA molecule, called messenger RNA (mRNA). mRNA communicates the genetic message found in DNA to other areas of the cell so protein production can occur. Like DNA, RNA is made up of nucleotide base pairs and a sugar-phosphate backbone.
During translation phase, mRNA travels to the ribosome, which is the cell’s machinery that assembles proteins based on the instructions contained in the mRNA. Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene “off”.
This is because mRNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA. This synthesized nucleic acid is termed an “anti-sense” oligonucleotide because its base sequence is complementary to the gene’s messenger RNA (mRNA), which is called the “sense” sequence (so that a sense segment of mRNA ” 5′-AAGGUC-3′ ” would be blocked by the anti-sense mRNA segment ” 3′-UUCCAG-5′ “).
How Antisense Works? 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 proteins. 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. 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.
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New developments in the chemistry of antisense give further hope to developing potent, long-lasting antisense therapies that are more conveniently adminstered. 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 backbone 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-generation drugs are composed of both RNA-like and DNA-like nucleotides, while first-generation drugs are entirely DNA-like. Because RNA hybridizes more tightly to RNA than to 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. 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. “0”Generation “1st”Generation “2nd”Generation Phosphodiester(P=O) Phosphorothioate(P=S) 2’MOE (modifiedP=S) Characteristics: Characteristics: Characteristics: (1)
Highly unstable to (1) Better stability to nucleases (1) Best stability to nucleases. but still degrades.nucleases. (2) Good affinity to target (2) Decreased affinity to target (2) Increased affinity to target mRNA mRNA mRNA Result: Result: Result: (1) Rapidly degrades (1) Unknown tissue concentration. (1) Confirmed tissue concentration (2) Never advances to ( 2) Unknown target regulation. (2) Confirmed target regulation human trials. (3) Daily dosing. (3) Once a week dosing RESEARCHES: Flavr Savr, a genetically modified tomato, was the first commercially grown genetically engineered food to be granted a license for human consumption.
It was produced by the Californian company Calgene, and submitted to the U. S. Food and Drug Administration (FDA) in 1992. On May 17, 1994, the FDA completed its evaluation of the Flavr Savr tomato and the use of APH(3′)II, concluding that the tomato “is as safe as tomatoes bred by conventional means” and “that the use of aminoglycoside 3′-phosphotransferase II is safe for use as a processing aid in the development of new varieties of tomato, oilseed rape, and cotton intended for food use. ”
It was first sold in 1994, and was only available for a few years before production ceased in 1997. Calgene made history, but mounting costs prevented the company from becoming profitable, and it was eventually acquired by Monsanto Company Antisense drugs are being researched to treat cancers (including lung cancer , colorectal carcinoma , pancreatic carcinoma , malignant glioma and malignant melanoma ), diabetes , Amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy and diseases such as asthma and arthritis with an inflammatory component.
Most potential therapies have not yet produced significant clinical results, though one antisense drug, fomivirsen (marketed as Vitravene), has been approved by the U. S. Food and Drug Administration (FDA) as a treatment for cytomegalovirus retinitis .
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%. 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 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.
Cancer Also in 2006, German physicians reported on a dose-escalation study for the compound AP 12009 (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 . Potential benefits; potential risks Gene technology promises increased yields and reduced dependence on pesticides. However, as well as potential benefits, there are also potential risks. For example, if an insecticide-producing gene inserted into a crop plant accidentally ‘escaped’ into a wild relative of the crop, then the wild relative might become a problem weed.