Antisense Oligonucleotide Therapies: Are They the “Magic Bullets”?

The recent commencement of clinical trials using antisense oligonucleotide therapy for leukemias and viral infections has heralded a new era in drug therapy [1-4]. Antisense oligonucleotides are short chains of nucleic acids, usually 10 to 30 residues long, and are intermediate in size compared with smaller-size conventional drugs, such as β-blockers, and the much larger therapeutic polypeptides, such as growth factors or monoclonal antibodies. Conventional drugs generally affect cellular functions by interacting with proteins.

Despite substantial advances in the understanding of molecular interactions between proteins and their ligands, it is difficult to design drugs based on the amino acid sequences of proteins because ligand-protein interactions involve multiple chemical bonds. In contrast, regardless of the gene function, antisense oligonucleotides can be designed based on the nucleotide sequences of the targeted genes and on the concept of base-pairing of nucleic acids, which is governed by one set of physical-chemical principles. The mechanism of recognition between two nucleic acid strands is through hydrogen bonding of the four nucleotide bases that make up the genetic alphabet. A properly designed antisense oligonucleotide, with a specified complementary base sequence, binds selectively to a targeted region of the messenger RNA (that is, the sense strand) and prevents RNA translation into protein. The specificity and stability of an oligonucleotide also depend on its length. The therapeutic aim is to inhibit genes that are vital to the survival or the function of the target cell or organism. Theoretically, these synthetic oligonucleotides can be more specific than most conventional drugs and can inhibit mutated genes or foreign genes without affecting the normal genes.

Oligonucleotide therapy is not confined to the antisense strategy [3]. Another approach is the antigene strategy whereby gene inhibition can be achieved through triplex formation between the synthetic oligonucleotide and the double-helical DNA. A further example is synthetic oligonucleotides designed to mimic the action of ribozymes, which are naturally occurring RNAs capable of degrading RNAs in the same manner that enzymes degrade proteins. Therapeutic nucleic acids are not confined to single-stranded nucleic acids and include double-stranded DNAs [3]. Gene therapy is also a rapidly developing field with the goal of permanently replacing a missing or deficient gene. Recently, DNA has also been used as a vaccine to elicit immune responses, as shown in mice [5].

There are several obstacles to antisense therapy [1, 2]. Antisense oligonucleotides with a natural phosphodiester backbone are degraded rapidly in serum and cross the cell membrane poorly. However, they can be modified to render them resistant to degradation by replacing the oxygen in the phosphate backbone with sulfur (phosphorothioates) or with methyl groups (methylphosphonates). Other chemical modifications have been developed to enhance the biologic effect of oligonucleotides. The use of carriers (such as liposomes, polymers, and retroviral vectors) has been investigated to improve the bioavailability of synthetic oligonucleotides [6].

Viruses are the obvious target for antisense therapy [7], as most are difficult to treat by conventional drugs. Because many viral genes differ substantially from human genes, higher target specificity and lower toxicity could be achieved using antisense therapy. The potential therapeutic effect of synthetic oligonucleotides was first published in 1978 and showed the inhibition of Rous sarcoma viral RNA translation and viral replication in cells [8]. Progress was hindered until advances occurred in the synthetic chemistry of oligonucleotides and in an increased understanding of the role of genes in disease pathogenesis.

In cell culture, growth of acyclovir-resistant herpes simplex [9] and hepatitis B viruses has been effectively blocked by antisense oligonucleotides alone or by oligonucleotides delivered using a DNA carrier [10]. Production of human immunodeficiency virus (HIV) in both acutely and chronically infected human cells can be inhibited by unmodified and modified antisense oligonucleotides. Attempts to block HIV integration into the human genome have been partially successful. A recent report [11] found that sequential in vitro treatment of HIV-1-infected cells with different oligonucleotides at a clinically achievable drug concentration prevented the emergence of resistant strains; this treatment might decrease viral burden in patients. A clinical trial using an anti-gag phosphorothioate in patients with the acquired immunodeficiency syndrome is now under way. In animal studies, topical and systemic applications of antisense oligonucleotides [7, 9] have had some success in treating cutaneous, ocular, and systemic herpetic infections. Recently, a human papilloma viral gene was inhibited by a 20-residue phosphorothioate oligomer, and a clinical trial has begun using this drug for genital warts [12]. The potential of this class of drugs to treat infectious diseases extends beyond viruses. Parasites such as Trypanosoma brucei[3], the culprit in African sleeping sickness, and chloroquine-resistant Plasmodium falciparum malaria [13] were also susceptible to growth inhibition by antisense drugs in vitro.

In cancers, a number of cellular genes (proto-oncogenes) are altered because of point mutation or chromosomal translocation. Selective inhibition by antisense oligonucleotides directed against several oncogenes has been shown [14]. In chronic myeloid leukemia, antisense oligonucleotides targeted to the mRNAs of the bcr-abl fusion gene or c-myb suppressed leukemic cell proliferation [15, 16]. Early clinical studies using these antisense oligonucleotides in chronic myeloid leukemia are now under way. In some tumors, the Ha-ras gene is activated by a single nucleotide mutation. Antisense oligomers can selectively inactivate the mutated but not the normal Ha-ras gene [14], showing the highly selective action of antisense oligonucleotides. Further, growth of these tumor cells is suppressed in vitro and in vivo [17]. The differential sensitivity of normal and cancer cells to antisense oligonucleotides shown in some laboratories needs to be substantiated.

This new class of pharmaceutical compounds could potentially be used to treat other diseases. Antisense oligonucleotides directed against neuropeptide genes alter behavioral patterns in animals [18]. Several groups have noted the antiproliferative action of synthetic oligonucleotides on vascular smooth muscle. Local application of an anti-c-myb oligonucleotide inhibited intimal smooth muscle cell accumulation in rat arteries subjected to balloon angioplasty [19], which may be a new approach for preventing post-surgical arterial restenosis.

Despite these exciting discoveries, many uncertainties remain about the therapeutic potential of synthetic oligonucleotides. These issues include the unequivocal demonstration of effectiveness and the specificity of antisense oligonucleotides in biological systems [20]. High concentrations of oligonucleotides (such as phosphorothioates) produce non-antisense-mediated inhibition. High production costs of large-scale manufacturing and pharmacologic issues, particularly efficient drug delivery systems, are problems that need to be addressed before antisense oligonucleotides can be used routinely [2, 6]. As with conventional drugs, repeated administration of oligonucleotides is needed to maintain the therapeutic effect. Various methods used for drug delivery are being applied to antisense therapy, such as topical application and intravenous infusion.

The pharmacokinetics and biodistribution of some of the first-generation oligonucleotides have been favorable [2, 9, 21]. Oligonucleotides are distributed evenly to most tissues, and the excretion route is mainly in the urine. No acute toxicity was seen with therapeutic doses given to animals or in a phase-I clinical study of systemic administration of an anti-p53 oligonucleotide in patients with acute leukemia [21]. Clinical trials of these first-generation oligonucleotides are being done in several centers, and these studies will provide information on their pharmacologic activity and an indication of the efficacy of this form of therapy. They will also help to identify potential problems of toxicity, mutagenicity, emergence of antibodies, and drug resistance. Single agents will probably not cure diseases that have multistep pathogenesis. Hence, these compounds may be best used with other treatment modalities.

Information obtained from the first-generation antisense oligonucleotides could provide further insights into improving their design and provide a clearer indication of the genes and diseases amenable to treatment by this class of drugs. Although these compounds hold great promise, it is premature at present to consider them as the elusive “magic bullets”.

• Copyright ©2004 by the American College of Physicians