Advances in Nucleic Acid Therapeutics

Sudhir Agrawal, Michael J Gait, Sudhir Agrawal, Michael J Gait

• Publisher: Royal Society of Chemistry
• Year: 2019
• ISBN:
  9781788017329
• Edition: 1
• Topic: Scienze biologiche
• Subtopic: Biochimica

The sequencing of the human genome and subsequent elucidation of the molecular pathways that are important in the pathology of disease have provided unprecedented opportunities for the development of new therapeutics. Nucleic acid-based drugs have emerged in recent years to yield extremely promising candidates for drug therapy to a wide range of diseases. Advances in Nucleic Acid Therapeutics is a comprehensive review of the latest advances in the field, covering the background of the development of nucleic acids for therapeutic purposes to the array of drug development approaches currently being pursued using antisense, RNAi, aptamer, immune modulatory and other synthetic oligonucleotides. Nucleic acid therapeutics is a field that has been continually innovating to meet the challenges of drug discovery and development; bringing contributions together from leaders at the forefront of progress, this book depicts the many approaches currently being pursued in both academia and industry. A go-to volume for medicinal chemists, Advances in Nucleic Acid Therapeutics provides a broad overview of techniques of contemporary interest in drug discovery.

CHAPTER 1

History and Development of Nucleotide Analogues in Nucleic Acids Drugs

Sudhir Agrawal^*a and Michael J. Gait^*b

^a Arnay Sciences LLC, Shrewsbury, MA 01545, USA

^b Medical Research Council, Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
^*E-mail: [email protected], [email protected]

The nucleic acid-based drug discovery approach is now being recognized as a major platform in addition to small molecules and peptide- or protein-based platforms. Advancement in nucleic acid based drugs has been aided by the chemistry of oligonucleotides and nucleic acids in providing drug-like properties. Early experience was gained with respect to the use of the chemistry of oligonucleotides for use as antisense agents. These chemical structure–activity relationship studies included modification of the backbone, heterocyclic bases and sugars, or combinations thereof. In this chapter we discuss early developments in the chemistry of nucleic acids that have guided the design and successful development of antisense drugs as well as nucleic acid-based platforms employing many other mechanisms, including aptamers, siRNA, exon skipping, ribozyme, microRNA and non-coding RNA and immune modulation.

1.1 Introduction

Forty years ago, Zamecnik and Stephenson proposed the therapeutic use of antisense oligonucleotides on the basis of their finding that Rous sarcoma virus (RSV) replication could be inhibited by a synthetic oligonucleotide complimentary to the RSV genome. ¹ This concept opened up a new approach to drug discovery, namely an oligonucleotide binding sequence-specifically via Watson–Crick base-pairing to a complementary target RNA.

Since then, continuous progress has been made towards realizing the potential of this novel scientific approach and this has led recently to the approval of five antisense drugs. While the underlying concept of antisense is very simple, a rigorous understanding of the chemistry of nucleic acids had to be developed for its use in humans. In this chapter we discuss the history of this chemistry of oligonucleotides in antisense and the lessons learned from preclinical studies and clinical trials that have guided the development in conferring drug-like properties.

In parallel to the development of antisense (see Chapters 2–4), the application of synthetic oligonucleotides as therapeutic agents has evolved into broad applications involving multiple modalities. These applications include ribozymes (see Chapter 18), small interfering RNA (siRNA, see Chapters 10, 11 and 12), microRNA (see Chapter 8), aptamers (see Chapters 15 and 16), non-coding RNA (see Chapter 9), splicing modulation (Chapter 6), targeting toxic repeats (Chapter 7), gene editing (Chapter 17), and immune modulations (see Chapters 5, 13 and 14).

The common feature of these applications is that drug candidates are composed of natural nucleosides or nucleoside analogues linked via phosphodiester or modified linkages.

1.2 The Antisense Concept

In 1976, RSV was the only purified virus for which a sufficient quantity was available for potential sequencing. Maxam and Gilbert sequenced this RNA virus and noted that both ends of the linear viral genome bore the same primary sequence and were in the same polarity. It occurred to Zamecnik that the new piece of DNA synthesized by reverse transcription at the 5′-end of this retrovirus might circularize and hybridize with the 3′-end. Thus he considered the possibility of inhibiting viral replication by adding a piece of synthetic DNA to the replication system to block the circularization step by hybridizing specifically with the 3′-end of the viral RNA in a competitive way.

This experiment led to startling observations, including the inhibition of new virus particles and the prevention of transformation of chick fibroblasts into sarcoma cells. In a cell-free system, translation of the Rous sarcoma viral message was also dramatically impaired. These observations were the first to show proof of the antisense concept. ^1,2

Not much further progress was made in the field up to 1985, primarily for three reasons. First, there was still widespread disbelief that oligonucleotides could enter eukaryotic cells. Second, there was very little DNA (or RNA) genomic sequence available for targeting by antisense, and third, efficient automated methodologies to synthesize oligonucleotides in sufficient quantities were only just beginning to become established.

1.3 Developments in Oligonucleotide Synthesis

Although the principle of solid-phase oligonucleotide synthesis was first introduced by Letsinger and Mahadevan in 1965, ³ development of more efficient methods of oligodeoxynucleotide (ODN) synthesis on solid support took place from 1975 in the Gait laboratory by the phosphodiester chemistry ⁴ and from 1979 by the phosphotriester method in the Itakura laboratory ⁵ and the Gait laboratory. ^6,7 These methods were superseded by the outstanding phosphoramidite chemistry of Caruthers and colleagues, ⁸ which was automated by Applied Biosystems and other companies. This transformed the ability of non-chemists to obtain ODNs for biological purposes.

1.4 Choices for Antisense Oligodeoxynucleotide Modifications

In the mid-1980s the discovery of human immunodeficiency virus 1 (HIV-1) and the availability of its RNA sequence led Zamecnik to employ an antisense approach in attempts to inhibit HIV replication. ² Antisense ODNs with phosphodiester linkages (PO-ASO, Figure 1.1) complementary to various regions of HIV-1 mRNA were synthesized using automated synthesizers. In HIV-1-infected cells, these PO-ASOs inhibited HIV-1 replication and suppressed expression of HIV-1 related markers. ⁹ Such experiments were carried out using primary human cells and cellular uptake of ODNs was not a limiting factor. In these studies a control PO-ASO showed minimal inhibition of HIV-1 replication, providing evidence of sequence-specific antisense activity. These studies re-established the potential application of antisense as a therapeutic approach.

Figure 1.1 Chemical structures of DNA and DNA analogues (A) phosphodiester oligodeoxynucleotide (PO-ODN), (B) phosphorothioate (PS-ODN), (C) Rp PS-ODN, (D) Sp PS-ODN.

1.4.1 Backbone Modifications

It was realized that the use of PO-ASOs would have limited therapeutic application, since these ASOs would be degraded rapidly in biological fluids. Soon, the focus of research shifted into identifying novel analogues of oligonucleotides that would have increased stability against nucleases and maintain the sequence-specific hybridization for use in antisense studies. The mechanisms of ASO activities (RNase H and steric blocking) are reviewed in Chapter 2. The characteristics required for a good ASO oligonucleotide are summarized in Figure 1.2.

Figure 1.2 Nucleic acid-based therapeutics include the use of synthetic oligonucleotides as drug candidates. These candidates have various characteristics including DNA or RNA of varying sequence composition, single- or double-stranded, formulated, conjugated or complexed with lipid carriers etc. While the intended target of these candidates is largely RNA there are innate immune receptors including TLR 3, 7, 8 and 9, RIG-1, STING and inflammasomes. These receptors are known to recognize patterns of nucleic acids and activate appropriate immune responses. Modulation of these receptors has shown broad therapeutic applications. In design of nucleic acid-based therapeutics and their intended mechanism of action due consideration is needed to avoid overlapping mechanisms.

1.4.1.1 Phosphorothioates

The ASO therapeutics field took a major step forward in the mid 1980s through the chemical synthesis of phosphorothioate (PS) ODNs, ¹⁰ based on the pioneering work of Eckstein. In these analogues a simple sulfur atom replaces an oxygen atom (Figure 1.1). PS-linked ODNs are much more resistant to nuclease degradation than phosphodiesters and thus cellular activities were found to be much higher. However, mixed diastereomeric PS-ODNs, accessible readily by automated synthesis, have lower binding affinity to target RNA as compared with PO-ODN. Synthetic methodologies were optimized to synthesize milligram quantities of PS-ODNs for use in experiments as ASOs.

Studies with PS-ASO targeted to various regions of HIV-1 mRNA as well as non-complementary analogs including homo-oligomers, were conducted in HIV-1-infected cell-based assays and showed potent dose-dependent inhibition of viral replication and antiviral activity. ^11,12 The antiviral activity was related to the base composition of the analogues, and longer PS oligonucleotides were more effective than shorter ones. These studies also established that in primary human cells, cellular uptake of PS-ASO was efficient and that no carrier was required. Furthermore, PS-ASOs also showed very potent and durable inhibition of HIV-1 replication in chronically HIV-1-infected cells. ^13,15

On the basis of the promise of these results, antisense technology gained the attention of the broader scientific community and PS-ASOs became the choice as first-generation ASOs. PS-ODNs thus quickly became the primary choice for therapeutics development by newly formed biotechnology companies, such as Gilead Sciences, Isis Pharmaceuticals (now Ionis), Hybridon (now Idera Pharmaceuticals) and several others. Over the next few years, hundreds of reports appeared in the literature on the use of PS-ASOs targeting various viruses, ^16,18 oncogenes, ^19,20 kinases ^19,21 and other targets. ²² Soon it was realized that cellular uptake in transformed cells in culture was poor and lipid-based formulations were needed for efficient uptake and antisense activity. Also it was noted that the duplex of a PS-ASO wi...