Revival: Delivery Strategies for Antisense Oligonucleotide Therapeutics (1995)

Saghir Akhtar, Saghir Akhtar

• Publisher: CRC Press
• Year: 2017
• ISBN:
  9781351366137
• Edition: 1
• Topic: Medicine
• Subtopic: Pharmacology

* The first book to focus solely on the delivery of antisense nucleic acids * Contributions from leading scientists in the antisense field * Review-type articles to introduce newcomers to the field * Review-type articles linked with exciting previously unpublished data on delivery of antisense therapeutics * Information on the state-of-the-art strategies for improved cellular and tissue delivery of antisense, antigene, and ribozyme nucleic acids

Chapter 1
Disrupting the Flow of Genetic Information with Antisense Oligodeoxynucleotides: Research and Therapeutic Applications

David Kregenow, Mariusz Z. Ratajczak, and Alan M. Gewirtz

Contents

1. I. Introduction
2. II. Molecular Interactions of Antisense Oligomers
3. III. Biochemistry of Antisense Oligomers
4. IV. Pharmacodynamics of Antisense ODN
5. V. The Trouble with Antisense
6. VI. Potential Applications of the Antisense Strategy in Clinical Medicine
7. VII. Perspectives on the Therapeutic Applications of Antisense DNA Oligomers in the Treatment of Human Leukemias — an Example of the Utilization of this Strategy in Clinical Medicine
8. VIII. Conclusions
9. References

I. Introduction

The ability to efficiently disrupt gene expression has become an important experimental approach for studying the biological function of a gene at the single-cell level and in intact organisms.^1-3 This capability also possesses promising pharmaceutical applications because of the potential utility of downregulating the expression of genes which contribute to disease states. The downregulation of over- or aberrantly expressed proto-oncogenes or oncogenes in malignant cells, for example, could inhibit cell proliferation, force cells to undergo apoptosis, or lead to reversion of the malignant phenotype. Inhibition of intimal or smooth muscle cell proliferation might be of use in the treatment of cardiovascular disease.^4-6 Finally, inhibiting viral and parasitic genes responsible for replication may be useful in the treatment of many infectious diseases.^7-12 No doubt many other applications await elucidation.

Relevant technologies for disrupting gene expression include homologous recombination, which, in taking advantage of natural crossover events that occur during cell division actually destroys the targeted gene^2,3 and the use of reverse complementary DNA^7,13-15 or RNA^14,15 to interfere with utilization of the mRNA of the target gene. These are the so-called "antisense strategies". Each of these approaches for manipulating gene expression relies on specific nucleotide base pairing for targeting the gene of interest, but only the antisense approaches are applicable in fully developed organisms. Accordingly, it is the antisense strategies which have received the most attention and development with regard to therapeutic application.

The antisense mRNA strategy relies on the transfection and subsequent expression of a plasmid carrying the cDNA of the gene of interest subcloned into the vector in an antisense orientation.^14,15 After transfection into the cell, the plasmid expresses the antisense mRNA which, because of its complementarity, is capable of hybridizing exclusively with the mRNA of the gene of interest. The mechanism of downregulation of sense mRNA by antisense mRNA mimics some physiological regulatory mechanisms observed in primitive organisms, where both strands of genomic DNA are transcribed, producing sense and antisense mRNA molecules.¹⁶ The sense-antisense mRNA hybrids are excluded from intracellular metabolism, and the translation of the mRNA is disturbed. Thus, gene expression is disrupted at the mRNA level. It is also possible to engineer into the antisense sequence a catalytic mRNA sequence which often increases the efficiency of mRNA destruction. Antisense RNA containing hammerhead or other catalytic sequences are called ribozymes.¹⁷

The antisense oligodeoxynucleotide (ODN) strategy relies on the introduction of short sequences of synthetic ODN into cells. Such ODN may be microinjected into cells or internalized by cells from the extracellular milieu. These ODN are able to hybridize within the cell to the appropriate DNA or mRNA sense sequence of the gene of interest.^1,7,13-15 Thus, DNA ODN can disrupt gene expression at the levels of transcription and translation. In addition, DNA ODN which show binding affinity to important intracellular proteins can be used as a means of selectively excluding these proteins from intracellular metabolism, thus downregulating gene expression at the protein level.¹³ The relative ease of synthesis and use of antisense ODN predict that they would be the preferred form of molecule for both research and therapeutic purposes. Accordingly, these molecules are the subject of intense scrutiny and experimentation. This review will focus on recent developments in this rapidly expanding area.

II. Molecular Interactions of Antisense Oligomers

In order to more fully understand how antisense ODN may perturb the flow of genetic information and thereby disrupt gene expression, it is necessary to review the process through which genes are expressed in living cells. Briefly, genetic information is encoded by the nucleotide sequence of chromosomal DNA. When a gene is to be expressed, it is transcribed from DNA into the corresponding nucleotide sequence of a mRNA molecule within the nucleus. Subsequently, the mRNA is processed, leaves the nucleus, and enters the cytoplasm. Here it is translated on ribosomes into the amino acid sequence of a protein. It is this protein which is responsible for producing the biological effect of the gene. Synthetic ODN can influence gene expression at each level — from DNA to protein (Figure 1).

First, under certain conditions, ODN can bind to sense sequences in the chromosomal DNA and create a triple helix.^13,19 This directly inhibits the transcription of the genetic information encoded by the DNA into mRNA.^21,22 The formation of a triple helix structure requires, however, that the targeted sense sequence within the DNA molecule contain a pyrimidine- or purine-rich motif.^13,20 The binding of ODN to such a motif follows the Hogsten's binding principle where thymidine binds to adeninethymidine pairs, creating a triplet (TAT) and protonated cytosine recognizes guaninecytosine pairs, creating another triplet (C⁺GC). According to this principle, a purinerich sequence on one strand of DNA such as AGAAAGGAGAAAAAGGGG will bind along the major grove of the double helix with synthetic ODN containing the sequence TC⁺TTTC⁺C⁺TC⁺TTTTTC⁺C⁺C⁺C⁺. The effectiveness of the antisense strategy to directly inhibit transcription is limited by the paucity of sufficiently long runs of pyrimidineor purine-rich motifs in the genomic sequence of interest to allow the formation of stable triple helices. In spite of this limitation, the formation of a triple helix has already been shown to inhibit the expression of several genes, and a number of novel approaches to overcome this technical constraint have also been used with success. For example, the antisense ODN can be designed with molecular links which hybridize to separate motifs on opposite strands.¹³ In addition, ODN conjugated with EDTA-Fe are able to generate free radicals and destroy the structure of a gene at the site of hybridization.¹⁹ This strategy has been employed successfully to cleave a part of the human fourth chromosome.²³

Figure 1 Potential sites at which ODN may disrupt gene expression. (1) Triple helix formation with chromosomal DNA disrupts transcription. (2) Hybridization to nuclear RNA disrupts RNA processing and/or (3) translocation to the cytoplasm. (4) Hybridization to mRNA disrupts the binding of ribosomes and/or (5) translation into protein. (6) Hybridization to mRNA provides a substrate for RNase H. (7) ODN interactions with proteins disrupt protein function.

The second potential interaction of ODN relies on the fact that antisense ODN can hybridize according to Watson-Crick base pairing inside the cell to the corresponding sense sequence in mRNA molecules and prevent their translation into protein. Synthetically created antisense ODN are designed against specific sense sequences in the mRNA molecules of the gene of interest.^24-28 Antisense ODN may impair translation after hybridizing to its corresponding mRNA sense sequence through a number of mechanisms: (1) hybridization to pre-mRNA in the nucleus may impair subsequent splicing and processing to mRNA; (2) hybridization can impair the translocation of mRNA from the nucleus to the cytoplasm; (3) hybridization can prevent the binding of ribosomes to the mRNA molecule; (4) hybridization can impair the translation process itself; and (5) the hybridized antisense oligomer-mRNA duplex is a substrate for intracellular digestion by RNase H.

The third potential point of oligomer interference with gene expression, also shown in Figure 1, relies on the ability of ODN to bind to proteins important in cellular homeostasis and thereby exclude them from metabolism.¹³ For example, this strategy has been shown to completely eliminate the actions of DNA regulatory proteins known as transcription factors.²⁹ The DNA regulatory proteins bind to short ODN containing the specific DNA sequence they recognize, leaving an insufficient number of protein molecules available to bind to the regulatory sequences in the gene promoter. The techniques for synthesizing ODN capable of binding to other biologically important proteins are still being refined...