Antisense therapy has been on a roller coaster ride since its inception. Is this heavyweight really down for the count?
“I get knocked down,
but I get up again,
You’re never going
to keep me down”
These lyrics from a song by Chumbawamba aptly describe the long odyssey of antisense therapy, which has had an eventful sojourn with its occasional set backs and timely regress. Since the time antisense was first described, the field has gone through numerous ups and downs. From the initial hype to downfalls, from large-scale production to in vivo rejections, from first product launch to rejection of New Drug Approval (NDA), antisense therapy has been an in-fashion, out-of-fashion field.
Nevertheless, the antisense principle is still an attractive concept and has retained its charm from the time it was first discovered. During the past few decades, much has been studied about the basic mechanisms of antisense, the medicinal chemistry, and the pharmacokinetic, pharmacologic, and toxicologic properties of antisense molecules.1-3 Moreover, discoveries in basic biology and disease mechanisms, coupled with advances in genomics, have fueled interest in the field of antisense therapy, leading to new-found enthusiasm.
The success of antisense technologies has been barricaded mainly due to the wide array of obstacles faced by antisense molecules to reach their site of action. However, the new-age developments in biological science, chemical modifications, and drug delivery developments have shown the promise of bright futures for antisense technologies.4, 5
How does it work?
To know the basic mechanism of antisense techniques, one needs to be familiar with the central dogma of molecular biology, which depicts the usual route of protein production from the basic genetic unit (Figure 1). The concept underlying antisense technology relies on the inhibition of the expression of a specific mRNA by the use of a complementary sequence, which leads to a blockage in the transfer of genetic information from DNA to protein.6
Antisense agents are complementary strands of small portions of “sense”, messenger RNA (mRNA), typically 15 to 25 bases long. When the antisense oligonucleotide hybridizes with its cognate mRNA target and forms a hybrid, the unnatural structure leads to the destruction of mRNA by RNase H, a cellular ribonuclease that degrades hybrid nucleic acids. Thus, the translation of the protein encoded by the target mRNA is inhibited.
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 and control the expression of unwanted proteins. The bound mRNA is marked for rapid breakdown by the cell’s enzymes (RNase H), thus freeing the antisense oligonucleotide to seek out and disable another identical mRNA strand.7
Hurdles on the path
The challenges encountered in developing applications of antisense technologies are numerous: enhanced stability, efficient delivery, identification of selective sites in the target RNAs, and minimization of off-target effects.
Nuclease attack: To inhibit translation, the antisense oligonucleotides must reach the interior of the cell unaltered. That ability depends upon stability of the oligonucleotide toward extra and intracellular enzymes. Combating nuclease attack is one of the prime requirements for maintaining the efficiency of antisense agents. Natural phosphodiester oligodeoxynucleotides (or oligoribonucleotides) are quickly digested by nucleases both in vitro and in vivo and thus the overall activity of antisense is compromised. A vast number of chemically-modified nucleotides have been used in antisense experiments; fortunately it has been found that the chemical modification usually helps antisense molecules circumvent nuclease digestion.4, 6, 10
Delivery issues: Despite the encouraging prospects of avoiding nuclease digestion through manipulation of nucleotide chemistry, an important hurdle that has to be overcome for successful antisense applications is cellular uptake of the molecules. The charged nature of oligonucleotides hampers their effective transfer through hydrophobic cellular membranes. A number of methods have therefore been developed for in vitro and in vivo delivery. By far the most commonly and successfully used delivery systems are nanoparticulate systems, which include liposomes and charged lipids that can either encapsulate nucleic acids within their aqueous center or form lipid–nucleic acid complexes as a result of opposing charges. These complexes are usually internalized by endocytosis.5, 8, 9
Targeting potential off-target effects: Finding an appropriate target site within the mRNA can be quite problematic for antisense agents. The limitations lie in identification of sense/antisense combinations that provide the most potent knock-down at the lowest possible concentrations. Selecting the target can be best done by systematically studying all the potential targets. Off-target effects refer to any non-sequence-specific effects caused by the antisense agents. Parameters such as the length of the molecule, conformational structure and thermodynamic considerations can give better insight into target-binding features and help in controlling off-target effects.8, 10
Side effects: Other potential issues in considering antisense technologies for application are unanticipated side effects. Antisense molecules are considered foreign objects by the body and thus they tend to elicit strong innate and acquired immune responses.6, 8
Antisense technologies have been hailed as one of the best options for gene manipulation to be used in studying gene function (functional genomics) and for discovering new and more specific treatments for a wide range of diseases in humans, animals, and plants (antisense therapeutics). However, this success has been mainly restricted to laboratory and research organizations. There has been only one antisense drug on 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 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.7, 11
About the Author
Ashok Patel is a senior research fellow at the Univeristy Institute of Chemical Technology in Mumbai, India.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 1, January, 2008, pp. 30-32.
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Filed Under: Drug Discovery