By Jennifer  Maxon, RN, and Charles H. Weaver, MD
January 2005 

Introduction

As the aim of cancer therapeutics is becoming streamlined to target more specific biological pathways, genetic components, and/or cellular proteins, the role of antisense therapy utilizing oligonucleotides is evolving as a potential treatment strategy in the fight against cancer. In the arena of scientific and medical research, antisense and/or oligonucleotide strategies may involve several strategic mechanisms of action.^1,2,3 However, the primary theme of antisense therapy currently being explored for oncology purposes is the inhibition of translation of a targeted protein through complementary oligonucleotide binding to target mRNA.^4,5,6 The obvious choices of deliberate inhibition through antisense therapy include specific proteins believed to be involved in the formation of malignancy, the continued expression of malignancy, and/or the resistance to therapy.

Mode of Action

At present, the main focus of antisense therapy in oncology involves the use of approximately 20 nucleotides (oligonucleotide) synthesized to be complementary to the specific “sense” (5’ to 3’orientation) mRNA sequence responsible for coding of the targeted protein.^7,5,6 Some of the more well-known proteins currently being targeted with antisense agents in development are Bcl-2, H-ras and PKC-Alpha (see Table 1). These proteins, as well as several others presently under investigation, have been implicated in playing a role in the development, growth, and/or maintenance of several types of cancer and tend to be involved in messaging systems involving anti-apoptotic signaling and/or unregulated cellular proliferation.⁷

Table 1: Antisense Agents and Their Targets

Once introduced into a cell, the “antisense” oligonucleotide hybridizes to the corresponding mRNA sequence through Watson-Crick binding, forming a heteroduplex.^8,9 Once the duplex is formed, translation of the protein coded by the sequence of bound mRNA is inhibited.10 There are several mechanisms through which the oligonucleotide/mRNA duplex may hinder subsequent translation.^6,7 The most widely accepted explanation for several different antisense agents involves the degradation of the mRNA in the heteroduplex by the ubiquitous enzyme Rnase H. Rnase H is attracted to the heteroduplex and cleaves the bound mRNA, while leaving the oligonucleotide sequence intact, allowing the oligonucleotide to continue seeking and binding to corresponding mRNA sequences.11 Some other accepted explanations of translation inhibition through antisense therapy which may occur separately or in conjunction with Rnase H activity include, but are not limited to, the blocking of appropriate ribosome assembly that disables the ribosomal complexes’ ability to translate, blocking of RNA splicing and/or impeding appropriate exportation of mRNA.^4,12,13,14

Potential Advantages

There are several aspects of antisense therapy utilizing oligonucleotides that are potentially advantageous over traditional drug mechanisms.

• Oligonucleotides may be manufactured quickly, some within one week, and the sequence of a gene is all that is needed.⁵
• Potential sensitivity to therapy may be easily measured, as the target is often one-dimensional versus multiple-dimensional domains often targeted within proteins. Sensitivity can be measured through database scanning or Northern/Southern blotting for unknown genes.¹⁵
• Potential to produce longer lasting responses, as clonal expansion may require more time to produce clinical disease once mRNA is inhibited, versus just inhibition of protein typical with conventional therapies.¹⁵
• Potential for enhanced binding affinity to target, as hydrogen bonding between oligonucleotide and target appears to exceed, by several orders of magnitude, Van der Waals and other forces used by standard agents to bind to protein targets.¹⁵

Structure

The concept of synthesizing a short strand of nucleotides complementary to a desired mRNA sequence appears fundamentally simple. However, several obstacles were recognized as this technique progressed through experimentation. One important hurdle that had to be addressed was the intrinsic nature of ubiquitous nucleases existent in living tissues and serum to degrade single-stranded nucleotide chains (i.e. the oligonucleotide). To overcome this initial impediment, a sulfur atom was substituted for the non-bridging oxygen in the nucleotide linkages of the synthesized chain. This chemical modification gives rise to a phosphorothioate backbone of the oligonucleotide that eludes immediate nuclease degradation while maintaining the ability to attract Rnase H.^16,9 The phosphorothioate modification is considered to be a first-generation antisense oligonucleotide, and is currently the most commonly seen modification in chemical structure in antisense therapy involved in clinical trials for oncology.⁷ In addition, oligonucleotides used for antisense therapy in oncology tend to be around 20 bases in length, as much longer oligonucleotides demonstrated poor permeation into cells and required involved carrier systems, and those much shorter in length are theoretically poor at recognizing a very specific RNA sequence, resulting in the potential binding of untargeted mRNA.

Future directions

Currently, research efforts in antisense strategies are aimed at understanding the degree of involvement of single genes, or combinations of genes, in the course of disease. The purpose of this important area of research is to isolate the most critical targets and identify the feasibility of using genes as targets for therapeutic purposes. Other issues being actively pursued include altering chemical structures of the oligonucleotides to provide greater resistance against degradation, enhance binding affinity to target, and improving pharmacokinetics. Improving these properties will, in theory, reduce possible side effects, increase efficacy, enable lower dosing, and provide an opportunity for oral dosing.^17,18 Future generation compounds include modifications to address these issues, such as composition of more RNA-like nucleotides versus DNA nucleotides, alkyl modifications at the 2’ position of the ribose, polyamide linkages replacing deoxyribose phosphate backbones, and 3’ amino groups to replace the 3’-hydroxyl group of the 2’-deoxyribose ring, to name a few.^{17,19,20,21,22} The ever-present issue of optimal drug delivery persists with antisense therapy, and research continues to refine delivery systems in order to achieve the goal of directing active treatment only to the target while sparing untargeted molecules, and maintaining active levels of the compound through the delivery process.^23,24,25

Role in Genomics

Antisense properties provide a fast and convenient platform for recognizing which genes are responsible for the production of particular proteins and their possible role in biological systems. This contribution to oncology is becoming even more important as genomic sequencing nears completion. The next step in the genomic venture is the knowledge expansion of functional genomics, or the task of determining which genes are involved in disease and which are feasible targets for therapeutic purposes (therapeutic function versus toxicity). The ability of antisense technology to provide ease of product synthesis, targeting of a single intended gene, and quick, reproducible laboratory results, will accelerate the maturation of functional genomics, and thus oncology therapeutics.¹⁷

Role in Clinical Use

Although no antisense agents have been approved for the treatment of cancer, their movement toward potential use in future clinical oncology settings is apparent. Perhaps the most well-known antisense agents in the development of oncology thus far are Genasense® and Affinitak®. There are, however, several other antisense agents currently in clinical trials for the treatment of various cancers.

Genasense®: Genasense® (oblimersen sodium) is an antisense agent that is targeted against the Bcl-2 protein. The Bcl-2 protein is overexpressed in several cancers and is implicated in anti-apoptotic pathways, resulting in resistance to anti-cancer therapy. The goal of treatment with Genasense® is to sensitize cancer cells to the effects of therapy, not to compete with different treatment agents. Genasense® has demonstrated synergy with several chemotherapy agents, radiation therapy and immunotherapy and is often administered a few days prior to standard therapies. Currently, Genasense® is being evaluated in over 20 clinical trials for the treatment of several cancers, including hematologic cancers and solid tumors. Furthermore, Genasense® has been evaluated, or is being evaluated in combination with the following agents: Gleevec®, Rituxan®, paclitaxel, Camptosar®, Fludara®, cyclophosphamide, Taxotere®, Mylotarg®, dexamethasone, and cytosine arabinoside.⁵

Recently, a new drug application (NDA) has been submitted to the FDA for Genasense® in treatment combination for metastatic melanoma. The NDA was based on recent results of a phase III trial directly comparing dacarbazine plus Genasense® to dacarbazine alone in the treatment of metastatic melanoma. This trial involved approximately 770 chemotherapy-naïve patients with metastatic melanoma who were randomly allocated to dacarbazine alone or in combination with Genasense®.

The group of patients who had been followed for at least 12 months (N=480) and were treated per protocol had median overall survival times of 10.1 months for those treated with the combination of Genasense®/dacarbazine compared to 8.1 months for those treated with dacarbazine alone. In the intent-to-treat population, the addition of Genasense® increased median progression-free survival (78 days versus 49 days) and anti-tumor response (11.7% versus 6.8%), compared to the patients treated with dacarbazine alone. Side effects were reportedly not severe and not unexpected.⁵

Affinitak™: Affinitak™ (formerly ISIS 3521, LY900003) is an antisense agent targeted against the protein PKC-alpha. PKC-alpha , a member of the PKC family, is often expressed in cancer cells. It has been associated with the promotion of growth, development and survival of malignant cells, and resistance to chemotherapy. Results from a recent phase III trial evaluating Affinitak™ in the treatment of chemotherapy-naïve patients with stages IIIB and IV NSCLC did not meet the goal of its primary endpoint – an overall survival benefit. This trial involved 616 patients who were randomly allocated to Paraplatin®/Taxol® with or without Affinitak™. The median survival was 10 months in the Affinitak™ arm, compared with 9.7 months in the chemotherapy only arm. However, among patients who finished the prescribed 6 cycles of chemotherapy (n=256), median survival was 17.3 months for patients treated with Affinitak™, compared to 14.4 months for those treated with chemotherapy only (p=.054). In addition, among all 616 patients, survival was statistically greater (p=.048) for patients treated with Affinitak™ using a stratified log-rank statistical analysis considering predefined variables. Incidence of severe toxicities was not greater in the Affinitak™ arm.²⁶

Conclusion

The use of antisense therapy in oncology holds promise, particularly as research progresses in refining its drug delivery methods, increasing its affinity and improving its specificity. Perhaps antisense therapy will provide greater benefit when used earlier in the course of disease; however, only future studies can evaluate this issue. The fact that an antisense agent has already been FDA approved for clinical use (Fomivirsen for CMV- retinitis in AIDS patients) demonstrates the possibility of its use in diseases such as cancer. As oncology therapeutics are clearing a path to include treatment modalities other than traditional chemotherapy and radiotherapy, antisense therapy appears to be a contestant in the realm of novel treatment approaches.

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