Stand and deliver – effective antisense delivery

Before we can fully take advantage of antisense therapeutics, we first need to effectively deliver the RNAi agent to its target. When it comes to oligonucleotides, says Dr Catherine McKeen, there are many challenges surrounding drug delivery – but with many strategies in development, the future looks bright Since the advent of antisense therapeutics in the 1980s, few oligonucleotide based drugs have obtained FDA approval. A few examples are Vitravene (Isis Pharmaceuticals), Mucagen (Eyetech) and Kynamro (Isis Pharmaceuticals) but there are many more currently in clinical trials. In 2013 the Danish Council for Research reported 109 RNA based therapeutics in the pipeline with 58 in clinical trials and with involvement from 34 pharmaceutical/biotechnology companies. Isis Pharmaceuticals alone has more than 30 oligonucleotide based drugs in their pipeline. This shows that although there was a drift away from investing in RNAi technology in 2010/2011 by some of the major pharmaceutical companies¹, this is still very much an area of interest. One of the main issues with using oligonucleotides as therapeutics is that the phosphate linkages are prone to endo and exonuclease degradation, most often in the endosome where the oligonucleotide is trapped during cellular uptake. Modification to the oligonucleotide can prevent the degradation process; the modified oligonucleotide is still trapped but is stable enough to exist until the endosome breaks down releasing it into the cytoplasm. Unmodified oligonucleotides are also highly charged therefore often require the use of a transfection agent to deliver these into cells. [caption id="attachment_40429" align="alignright" width="200"] Figure 1: Common Sugar-Phosphate Backbone Modifications used in Oligonucleotide Therapeutics. Click to enlarge[/caption] As with any molecular therapy, delivery to the cell is an issue. Researchers involved in small molecule therapeutics have tackled this issue with the development of prodrugs^2,3_{. ­}The same principles have been applied to oligonucleotide therapeutics^4-7 although this is not the only method utilised. In this case, there are three categories; phosphate modification⁵, 5’-esterification⁴ and a carrier (delivery reagent) conjugated to the oligonucleotide via a cleavable linkage such as a disulphide bridge⁷. Once in the cell the protection or the carrier is removed from the oligonucleotide drug. For all other techniques, the carrier is retained although may become involved in some biochemical processes or hydrophobic interactions with the cell membrane. It is these processes that make the carriers effective delivery reagents. There are many modifiers developed as potential delivery reagents when conjugated to an oligonucleotide. Although far from complete, the more common types of modifiers are discussed. Figure 1 shows examples of the vast number of sugar-phosphate modifications applied to oligonucleotide therapeutics. While these were developed to improve nuclease resistance rather than to improve delivery, in some cases delivery was also enhanced. For instance one of the first modifications was to replace phosphate linkages with phosphorothioate linkages and in fact Vitravene has only phosphorothioate linkages. In addition to significantly increasing nuclease resistance, phosphorothioate oligonucleotides show reasonably efficient cellular uptake in some cells via receptor mediated endocytosis where the oligonucleotide interacts with receptors on the cell surface. [caption id="attachment_40430" align="alignleft" width="200"] Figure 2: Diastereomers of Methyl Phosphonate and Phosphorothioate Oligonucleotides. Click to enlarge[/caption] Although phosphorothioate oligonucleotides have an antisense effect due to sequence-specific interactions, non-specific protein interactions are known to occur^8,9 and show low binding affinity towards RNA. Phosphorothioate and methyl phosphonate oligonucleotides exist as diastereomers (see Figure 2). While for small molecule therapeutics, there would be a requirement to separate the components of such a complex mixture, to date a similar requirement for oligonucleotide based drugs does not exist since it is thought that any non-active oligonucleotide will be degraded in the cell. Methyl phosphonates are highly stable in cells but the neutral charge inhibits cellular uptake¹⁰. The same can be said of PNA, alkylphosphate and morpholino oligonucleotides but these have all shown promise as potential therapeutic agents. In fact morpholino derived oligonucleotides are currently in clinical trials showing activity against Duchenne muscular dystrophy (DMD) ¹¹. In this case, such backbone modified oligonucleotides are conjugated to a delivery agent resulting in enhanced cellular uptake. [caption id="attachment_40431" align="alignright" width="200"] Figure 3: Commercially Available Cholesterol Synthesis Reagents. Click to enlarge[/caption] The area of developing carriers (delivery reagents) for conjugation to oligonucleotides is vast and a few of the most common carrier types are outlined below. Lipid/Fatty Acid/Hydrophobic Based The hydrophobic nature of lipid type molecules is thought to form a micelle encapsulating the charged oligonucleotide. The lipid layer then interacts with the cell membrane allowing the oligonucleotide to pass into the cell. There are many examples of lipid and lipid type molecules that have been used for this purpose. Some of which are given below. Liposomes Liposomes are synthetic vesicles made up of a double phospholipid membrane which encapsulates an aqueous solution allowing this to pass through the phospholipid bilayer of a cell membrane. These are often used as transfection reagents with in vitro assays e.g. lipofectamine and although these are useful they can rarely be applied to in vivo assays. Cationic Lipids As previously mentioned one issue in using oligonucleotides as therapeutics is the negative charge arising from the sugar-phosphate backbone. The use of cationic lipids such as spermine¹² has been shown to improve cellular uptake where the polycationic ligand is thought to neutralise the negative charge on the oligonucleotide. In addition to improving cell delivery, this improves the hybridisation properties of the oligonucleotide when binding to the target. One issue in using this approach is that the solubility of the oligonucleotide conjugates in aqueous media decreases as the length of the cationic ligand increases. Cholesterol Cholesteryl-oligonucleotide conjugates have proved an efficient means in improving cellular uptake^13-16. This is the most commonly used lipid based delivery agent conjugated to an oligonucleotide. It was first introduced by Letsinger¹² at the 3’-end of an oligonucleotide using H-phosphonate chemistry.  Since then, several modifiers have been developed for introduction of a cholesterol modifier into an oligonucleotide using phosphoramidite chemistry, the most commonly used synthesis reagents are shown in Figure 3 although many others have been reported. As with the use of cationic ligands, the aqueous solubility of the oligonucleotide-cholesterol conjugate is markedly reduced. [caption id="attachment_40432" align="alignleft" width="200"] Figure 4: Commercially Available Palmitic Acid Synthesis Reagents. Click to enlarge[/caption] Palmitic Acid Palmitic acid is the most commonly utilised fatty acid found in the body. It is the first such acid produced during fatty acid synthesis and is the precursor to all others. This is known to conjugate to cysteine residues in proteins, in particular membrane proteins and is thought to aid the movement of proteins between compartments in the cell. Similarly, palmitic acid has been conjugated to BSA which can then be absorbed and utilised by the cell. Oligo-3’-palmitate conjugates have been shown to be potential telomerase inhibitors¹⁷. Examples of commercially available palmitic acid derived synthesis reagents are shown in Figure 4. Nanoparticles Ideally, the use of nanoparticles to enhance cellular uptake by the target cells will result in a reduction of toxicity compared to using the free drug alone. However, there is some evidence that the nanoparticles can be as hazardous as the free drug¹⁸. Various forms of nanoparticles have been utilised e.g. gold, silver, polymers. Typical polymer based nanoparticles include polyacrylates such as polyalkylcyanoacrylate¹⁹ (PACA) which are often used in conjunction with cationic co-polymers such as DEAE dextran²⁰. Here the co-polymer acts in a similar manner to a cationic lipid neutralising the negatively charged oligonucleotide. Gold nanoparticles are the most well-known and have been applied to many oligonucleotide applications including therapeutics²¹. Here the oligonucleotide is thiol modified to allow attachment to the gold surface e.g. Spherical Nucleic Acids (SNA)²². More recently these gold oligonucleotide nanoparticles have been used as the delivery reagent for Pt(IV) warheads²³. In this case the oligonucleotide is not the therapeutic but is part of the delivery system. Cell-Penetrating Peptides (CPPs) These were first reported in the late 1980s²⁴ and there are many reported examples where these have been used as oligonucleotide carriers²⁵. CPPs have proved especially useful in the delivery of charge neutral oligonucleotides such as PNA²⁶and morpholinos^11,27. Vitamins The use of vitamins as delivery agents has been shown to have potential since these are used by, rather than produced by, the target cells and hence are recognised²⁸. They are thought to be internalised by cells only after interaction with a binding protein and therefore have the potential for targeting of a specific cell type. To date retinol²⁹, folic acid³⁰, tocopherol³¹ and niacin³² have all been reported as potential carriers. At Link we believe that vitamins have an important role in oligonucleotide therapeutics not only in terms of cellular uptake but also by using a combination of vitamins, possibly in conjunction with an additional carrier, it may be possible to target particular cell types. It is not unusual to use a combination of carriers. For instance cholesterol has been used in conjunction with spermine³³. [caption id="attachment_40433" align="alignright" width="200"] Figure 5: Commercially Available Vitamin Based Synthesis Reagents. Click to enlarge[/caption] Commercially available vitamin products are shown in Figure 5. 5’-Niacin CE Phosphoramidite is a very new product still in the development stage, hence it is possible that the means of conjugation to the oligonucleotide may change as more data is acquired. This article only touches on the vast number of molecules conjugated to oligonucleotides as potential carriers/delivery reagents. 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New Potential Oligonucleotide Delivery Reagents, C.M.McKeen, S.Aitken, G.McGeoch, K.V.Morris & H.Yoo, Poster presentation, OTS 2013. 33. Cholesterol conjugated spermine as a delivery modality of antisense Oligonucleotide, Y.K.Im, M.S.Kim & H.Yoo, Int. J. Oral Biol., 2013, 38(4), 155-160. 34. a) Targeted Delivery Systems for Oligonucleotide Therapeutics, B.Yu, X.Zhao, L.J.Lee & R.J.Lee, AAPS J., 2009, 11(1), 195-203. b) The Chemistry and Biology of Oligonucleotide Conjugates, R.L.Juliano, X.Ming & O.Nakagawa, Acc. Chem. Res., 2012, 45(7), 1067-1076. Author Dr Catherine McKeen, Head of Technology Commercialisation, Link Technologies Ltd