Novel non-endocytic delivery of antisense oligonucleotides

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Abstract

Antisense oligonucleotides (ONs) have several properties that make them attractive as therapeutic agents. Hybridization of antisense ONs to their complementary nucleic acid sequences by Watson–Crick base pairing is a highly selective and efficient process. Design of therapeutic antisense agents can be made more rationally as compared to most traditional drugs, i.e., they can be designed on the basis of target RNA sequences and their secondary structures. Despite these advantages, the design and use of antisense ONs as therapeutic agents are still faced with several obstacles. One major obstacle is their inefficient cellular uptake and poor accessibility to target sites. In this article, we will discuss key barriers affecting ON delivery and approaches to overcome these barriers. Current methods of ON delivery will be reviewed with an emphasis on novel non-endocytic methods of delivery. ONs are taken up by cells via an endocytic process. The process of ON release from endosomes is a very inefficient process and, hence, ONs end up being degraded in the endosomes. Thus, ONs do not reach their intended site of action in the cytoplasm or nucleus. Delivery systems ensuring a cytoplasmic delivery of ONs have the potential to increase the amount of ON reaching the target. Here, we shall examine various ON delivery methods that bypass the endosomal pathway. The advantages and disadvantages of these methods compared to other existing methods of ON delivery will be discussed.

Introduction

Knowledge of the transport barrier and cellular delivery of antisense oligonucleotides (ONs) is of importance in the development of therapeutically effective ONs. If the drug molecule does not reach its intended target, then it cannot exert any therapeutic effect and may even cause non-specific effects if it interacts with the wrong target. Because the target for any antisense ON is in either the cytoplasm or nucleus, its activity would therefore depend on how well the molecule can penetrate the cell membrane and reach the target. As a general rule, the plasma membrane of living cells is relatively lipophilic and restricts the transport of large and charged molecules. Therefore, it might be expected that this membrane would also act as a barrier for polyanionic molecules such as ONs. Antisense ONs are typically 10–25 nucleotides long and thus have molecular masses that range from 3000 to 7500 daltons. Despite such relatively large size, antisense ONs have been reported to enter cells and exert their action. On the other hand, several investigators have reported that ONs do not significantly permeate cell membranes and that, in the absence of appropriate delivery systems, e.g., liposomes, they do not exhibit antisense activity. As we will see from the discussion, the problem of cellular delivery of ONs involves not only cellular uptake but also their intracellular distribution and availability at the target sites. This is due to the fact that ONs have a tendency to be trapped in specific intracellular compartments, e.g., endosomes and lysosomes and, therefore, their availability at the target sites may be very limited. It is therefore necessary to understand their uptake mechanisms and intracellular distribution in order to design more efficient antisense agents. In this article, we will first discuss the current stage of understanding of cellular internalization of ONs and their intracellular trafficking. Approaches to improve cellular delivery and intracellular target accessibility will then be followed.

Section snippets

Mechanism of cellular oligonucleotide uptake

Cellular uptake of ONs depends on several factors, including the chemical nature, size and sequence of ONs, cell type and density, and the experimental conditions used for the uptake studies [1], [2]. In most cases, ON uptake is relatively rapid, with a plateau resulting within 15–120 min following the addition of ONs. Uptake of ONs occurs via an energy-dependent process and can be inhibited by treating the cells with metabolic inhibitors or by lowering the temperature [3], [4], [5], [6], [7].

General approaches to enhance oligonucleotide uptake

Over the years, several approaches have been developed to improve cellular uptake of ONs. These include inclusion of ONs into liposomes or attaching them covalently or electrostatically to specific or nonspecific carriers. Some of these methods are summarized below.

Need for non-endocytic delivery

In order for the antisense ONs to be therapeutically effective, appropriate drug concentrations must be attained and maintained at the site of drug action. Various studies have unequivocally indicated that, once internalized, ONs are predominantly located in intracellular vesicles [45], where they are still separated by a membrane from their target [4], [45]. ONs thus tend to remain trapped inside endocytic vesicles and end up being degraded in the lysosomes (see Fig. 1). The efficiency of

Microinjection

Microinjection is a rapid way to screen and evaluate the inhibitory properties of antisense ONs. This is because the ONs can be directly injected into the cytoplasm [47] or nucleus [48] of an individual cell. Cells are cultured on glass coverslips on an inverted microscope and each cell is injected with the ON using glass capillary pipettes prepared using a needle puller [49].

Leonetti et al. [47] reported a rapid and preferential accumulation of microinjected ONs in the nucleus of intact cells.

Non-invasive non-endocytic delivery

In this section, we discuss a number of peptides that have either been used or have the potential to be used for the non-invasive delivery of ONs. Most of these peptides have membrane-translocating properties that can be exploited for the delivery of ONs.

Conclusion

Since the efficacy and clinical prospects of antisense ONs depend on the cytosolic or nuclear concentrations of the drug achievable, the means of improving cellular uptake and cytoplasmic/nuclear entry are of particular importance. In the past few years, there has been significant progress in this direction. Most studies to date suggest that ONs in the cytoplasm migrate rapidly to the nucleus, suggesting that the endocytic barrier poses a significant barrier to the antisense activity. Indeed,

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