Antisense Oligonucleotide

This intriguing area started in 1970s targeting viruses, and then moved to humans to target DNA. The basic idea of this novel therapeutic approach is to synthesize a short oligonucleotide complementary to a sequence of a certain gene. Hybridization ensues, preventing expression and replication of that DNA. However several problems arose including low hybridization and non-specificity.

So with the start of the 1990s, a new approach was taken. Instead of killing the message, let's kill the messenger, mRNA.

Many successes and failures followed and the current state of the technology is that several criteria are required to assure specificity, lower side effects and optimize delivery. It appears that these agents will find many medical applications in the next few years.

What's in a name?

The message (mRNA) is the correct nucleotide sequence that encodes for a protein. It is thus said to make 'sense'. The complementary sequence is thus termed 'antisense'.

Mechanism of Action:

Blocking translation by rendering the mRNA unavailable for translation. Recent studies have shown that a more important mechanism involves a DNA-RNA hybrid that activates an enzyme, RNase H that breaks RNA strands in any RNA-DNA duplex. 

Target:

Disease states that are associated with inappropriate production or overproduction of gene products.

Advantages:

The allure of antisense oligonucleotides lies in its potential specificity, where we can target selectively the pathogenic gene. If the sequence of the gene or mRNA is known, drug design is a straight forward process. No need for laborious and tedious experiments and SARs. They are generally easy to synthesize. A small amount needs to reach the targeted cells, as mRNA is present in minute quantities.

Potential Problem:

Specificity.

Inhibiting normal cellular processes.

Susceptibility to nucleases.

Non-specific binding.

Pharmacokinetics problems; oral formulation and cellular uptake.

Specificity:

The oligonucleotide needs to be around a 17-20-mer (17-20 nucleotides) for specificity (the probability of the same sequence is one in a trillion). It has to target an area that is accessible for hybridization. The earlier oligonucleotides targeted the initiation code, but it turns out that this is not always accessible. Although some software can help eliminate certain fragments, the process usually is done by trial and error. This specificity gives these agents a tremendous advantage, this low toxicity. In some cases it would be optimal to individualize the treatment. For example if you are targeting a mutated mRNA of the c-myc in cancer patients, you may interfere with the normal cellular processes. To make these agents specific you may need to uncover the exact mutation that occurred to the c-myc and direct the antisense oligonucleotide to that particular region [remember that the exact mutation may vary from one patient to another].

Attack of Nucleases:

Problems associated with the degradation of oligonucleotides by nucleases can be reduced by a simple change to of an O to an S, resulting in phosphorothioate oligonucleotides (PS) that are stable versus nucleases. These agents are the mainstream agents to be utilized and are sometimes called first generation antisense oligonucleotides.

Non specific Binding:

The major problems associated with these oligonucleotides are due to non-specific binding, which may lead to undesired effects. One potential problem is the presence of certain sequences, such as four successive Gs that may have unwanted effects especially on the immune system. Another problem arises from secondary structures that can take place due to hybridization of the oligonucleotide with it self. This will be followed by hybridization of the remaining sequence to nonspecific targets and causing unwanted effects. The presence of polyanionic moieties leads to non specific binding to proteins and other cellular components. The effect of such binding is variable and may range from immunostimulation to induction of cell proliferation to inhibition of cell proliferation. This can be overcome by the use of Methyl Phosphonates. However the presence of anionic portions is essential to induce and activate the RNase H. So mixed backbone oligonucleotides (second generation oligonucleotides), that contains both Methyl Phosphonates and Phosphorothioates are being investigated.

Pharmacokinetics:

They cannot be used orally as of yet, but research focused on this aspect is advancing. They are used by IV infusions, are well distributed. It can be given every other day on an outpatient basis; platelet counting is necessary, with the second-generation oligonucleotides alleviating toxicity problems. Cellular entry is usually low, as they are charged molecules that appear to enter the cells through an inefficient endocytosis mechanism. Thus the use of carriers such as liposomes appears to improve their cell entry.

Uses of Oligonucleotides:

Fomivirsen (Vitravene^®, ISIS 2922) is a 21-mer phosphorothioate directed against AIDS-related CMV that can induce retinitis.

Trecovirsen is a 25-mer phosphorothioate directed at the mRNA of the gag gene of HIV. The Phosphorthiote analog showed extensive toxicity and has been replaced by the second generation analog in clinical trials.

Target GFs and other proteins that may play role in cell proliferation. Clinical trials are underway for several agents:

• ISIS 5132 that targets kinases of c-Raf, and is being tested in lung, bladder and breast cancer cells.
• CGP64128A is a PS against PKC-α and is being tested in many cancers.
• Oligonucleotides against Bcl-2 in lymphomas are promising especially in non-responsive lymphomas.
• Oligonucleotides targeting Mdm-2 leads to restoration of p53 and induces apoptosis.

ISIS 2302 a 20-mer phosphorothioate directed against human intercellular adhesion molecule I (ICAM-1). It leads to inhibition of expression of this protein and shows anti-inflammatory activity in transplant rejections, Rheumatoid Arthritis and ulcerative colitis. Oligonucleotides directed against human Angiotensinogen are being tested in hypertensive patients.

New approaches:

Oligonucleotides targeting DNA. They will form a Triplex formation with DNA and inhibit replication. Oligonucleotides targeting proteins (Aptamers) have been isolated from various cells. Examples include Oligonucleotides binding to RNA Polymerase of Influenza virus, Reverse Transcriptase of HIV and those targeting thrombin (could be effective as an anticoagulant agent).

Ribozymes are RNA molecules that have a tertiary structure and can degrade RNA. They are being investigated against various disease states and viruses. They will target a specific mRNA by flanking the ribozymes with an antisense oligonucleotide specific to that mRNA.

Antisense oligonucleotides that modulate alternative gene splicing. An estimated 60% of all human genes undergo alternative splicing that produces splice variants with different functions. Such variants have been linked to a variety of cancers, and genetic diseases such as cystic fibrosis. The requirements for such oligonucleotides are different from those described, as they must not activate RNase H and effectively access the target pre-mRNAs. Methylphosphonates coupled with modifications to the bases, to increase the affinity of the oligonucleotides to their target, may facilitate favorable antisense activity in the context of splicing.

To listen to the lecture, click on one of the following links:

mp3^*:   Lecture