Current Cancer Drug Targets - Volume 1, Issue 3, 2001

Recent progress made in molecular biology, biotechnology, and genetics, especially in identifying, cloning, sequencing and characterization of normal and pathogenic genes, has led to the development of genetic therapy. Major efforts in the field can be summarized in two general approaches: gene therapy and antisense therapy. The second is to deliver to the target cells antisense molecules that target to mRNA with which they can hybridize and specifically inhibit the expression of pathogenic genes. Antisense oligonucleotides offer the possibility of specific, rational, genetic-based therapeutics. With encouraging results from preclinical and clinical studies of antisense oligonucleotides in the past decade, significant progress has been made in developing antisense therapy, with the first antisense drug now being approved for clinical use. In this article, we will discuss approaches to developing these drugs from preclinical to clinical settings. Of particular interest for the area of human cancer therapy, several cancer targets, including bcl-2, BCR-ABL, C-raf-1, Ha-ras, c-myc, PKC, PKA, p53 and MDM2, are reviewed as examples to illustrate the progress in this field and emphasize the importance of target selection and advanced antisense chemistry in the development of antisense therapy.

Antisense technology, which is based on a simple and rational principle of Watson-Crick complementary base pairing of a short oligonucleotide with the targeted mRNA to downregulate the disease-causing gene product, has progressed tremendously in the last two decades. Antisense oligonucleotides targeted to a number of cancer-causing genes are being evaluated in human clinical trials. While the first-generation phosphorothioate antisense oligonucleotides are in clinical trials, a number of factors, including sequence motifs that could lead to unwanted mechanisms of action and side effects, have been identified. The severity of the side effects of first-generation antisense oligonucleotides is mostly dependent on the presence of certain sequence motifs, such as CpG dinucleotides. A number of second-generation chemical modifications have been proposed to overcome the limitations of the first-generation antisense oligonucleotides. The safety and efficacy of several second-generation mixed-backbone antisense oligonucleotides are being evaluated in clinical trials. The immune stimulation affects observed with CpG-containing antisense oligonucleotides are being exploited as a novel therapeutic modality, with several CpG oligonucleotides being evaluated in clinical trials. A number of medicinal chemistry studies performed to date suggest that the immunomodulatory activity of CpG oligonucleotides can be fine-tuned by site-specific incorporation of chemical modifications in order to design disease-specific oligonucleotide therapeutics

It has been estimated that greater than 35percent of all human genes undergo alternative splicing. The process of alternative splicing is highly regulated and disruption of a splicing pattern can produce splice variants that have different functions. Certain splice variants that are associated with induction of cell death, regulation of cellular proliferation and differentiation, cell signaling, and angiogenesis are present in a variety of cancers. Several of these cancer-related alternatively spliced genes will be discussed in this review. In addition, alternative splicing is associated with several genetic disorders such as beta-thalassemia, cystic fibrosis, and muscular dystrophy. Control of pre-mRNA splicing patterns with antisense oligonucleotides presents an attractive way to potentially treat and manage a variety of diseases. This review will discuss potential gene targets for antisense oligonucleotide induced modification of alternative splicing patterns. Furthermore, the chemistries and delivery strategies of antisense oligonucleotides will be discussed.

Aberrant gene expression is characteristic to all cancer cells and pathophysiology in general. Selective inhibition of constitutively elevated expression of oncogenes provides an opportunity to hinder the proliferation of malignant cells. Small synthetic molecules that specifically interfere with transcription and / or translation have great potential as anticancer drugs. Currently first-generation antisense oligonucleotides are widely used to inhibit the oncogene expression. The second generation of antisense agents have been studied mainly in vitro. One of these agents, peptide nucleic acid (PNA) is an oligonucleotide mimic with a non-charged achiral polyamide backbone to which the nucleobases are linked. PNA oligomers bind tightly to complementary DNA or RNA and are very stable in biological fluids. PNA can inhibit transcription and translation of target genes by specifically hybridizing to DNA or mRNA. The in vitro experiments showing inhibition of target protein expression by PNA have been followed by the first successful applications of PNA as an antisense agent in cultured cells and also in vivo. Hopefully this will lead to a wider use of PNA in the studies of cancer biology and therapy.

D-RNAi (Messenger RNA-antisense DNA interference), a novel posttranscriptional phenomenon of silencing gene expression by transfection of mRNA-aDNA hybrids, was originally observed in the effects of bcl-2 on phorbol ester-induced apoptosis in human prostate cancer LNCaP cells. This phenomenon was also demonstrated in chicken embryos and a human CD4+ T cell line, H9. The in vivo transduction of beta-catenin D-RNAi was shown to knock out more than 99 percent endogenous beta-catenin gene expression, while the in cell transfection of HIV-1 D-RNAi homolog rejected viral gene replication completely. D-RNAi was found to have long-term gene knockout effects resulting from a posttranscriptional gene silencing mechanism that may involve the homologous recombination between intracellular mRNA and the mRNA components of a D-RNAi construct. These findings provide a potential intracellular defense system against cancer and viral infections.