Exon skipping is one of a number of “steric blocking” applications of oligonucleotides (ONs) and their analogues that in recent years have undergone a renaissance and which have led to new therapeutic opportunities (Kurreck, 2008; Kole et al., 2012; Lightfoot and Hall, 2012). Whereas most standard antisense applications involve cleavage of the RNA by intracellular ribonuclease H (RNase H) or by argonaute2 (Ago2) enzymes, exon skipping by steric blocking merely requires binding of the ON in an antisense orientation to an RNA target and blockage of the important biological function of splicing. In exon skipping, as well as the similar splice-switching activity of exon inclusion, the target is pre-mRNA located in the cell nucleus. Thus, the ON must enter the cell nucleus in sufficient quantity to be in excess over the target pre-mRNA, bind to it strongly, and interfere with the splicing machinery. For drug use, the ON must exhibit additionally a number of other important properties such as good biodistribution to the target organ(s), lack of immune recognition effects, and a good therapeutic window between effective and toxic doses. Further, ON synthesis must be routinely achievable on varying scales. Such simultaneous requirements have taxed the ingenuity of chemists in ON design. It is necessary in all therapeutic applications of ONs to include modifications to the ON backbone and/or sugar component to protect against nuclease degradation. Interestingly, the very first analogue to find use therapeutically was the phosphorothioate linkage (Matsukura et al., 1987), which is still utilized in most ONs in current clinical trials. Further modifications have followed in subsequent years that have improved stability to nucleases, or even result in the ON being nuclease inert. The best ON analogues both enhance RNA binding strength, compared with an unmodified ON, and reduce nuclease resistance. Increased binding strength has allowed shorter ONs to be used in some cases, thus reducing the chance of binding to an incorrect RNA site through partial sequence complementarity. For example, in the case of ONs containing locked nucleic acids (LNAs), lengths of 12 to 15 are usually used (Lanford et al., 2010; Straarup et al., 2010) and even shorter in the case of all-LNA ONs (Obad et al., 2011). More commonly, lengths of ON analogue synthesized for exon skipping and other steric blocking applications are 14–30 residues. ONs are usually taken up into cells via endocytosis and then they must be released sufficiently from endosomal compartments into the cytosol. The cell nucleus is not thought to be a barrier for ONs once released from endosomes. However, cell entry behavior in culture is not a good predictor of in vivo activity. Thus for a particular exon skipping application, once in vivo delivered, the ON must be able to reach the required cell types. Leading applications for exon skipping and exon inclusion have been neuromuscular diseases such as Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). In both these cases, as well as in targeting of triplet repeats in myotonic dystrophy, the ON must be able to penetrate muscles of various types. However, it has proved very hard to date to find ONs or derivatives that are, in addition to skeletal muscle, as effective in heart and/or brain. Further, parameters such as biodistribution and organ accumulation, toxicology, and pharmacology in mice or rats must each be assessed, thus requiring at least multimilligram quantities of the ON to be readily obtainable. For such studies in animals and for clinical trials, the ON must be synthesized easily and cost effectively on gram to kilo scale under good manufacturing practice (GMP). Only a very small number of splice switching ON analogues have advanced sufficiently for use in clinical trials (Muntoni and Wood, 2011; Kole et al., 2012; van Deutekom et al., 2013) but some newer analogue types appear promising. The chemistry types that have been under study for exon skipping are depicted in Fig. 1 and the applications for which they have been used are shown in Table 1. FIG. 1. Structures of repeating units of analogues of oligonucleotides (ONs) used in exon skipping and related applications. (R=O or S) (A) Charge-negative ONs. (B) Charge-neutral ONs. Table 1. Oligonucleotide Types and Their Exon Skipping, Exon Inclusion, and Related Applications Negatively Charged Oligonucleotide Analogues A 31-mer oligodeoxynucleotide phosphorothioate (DNA-PS, Fig. 1A) was the first ON analogue to make it to a clinical trial in a single patient for exon skipping in DMD (Takeshima et al., 2006). A disadvantage of DNA-PS is that when bound to RNA it can induce undesirable cleavage of the RNA by cellular RNase H. Very few other types of negatively charged ON analogue that do not induce RNase H cleavage have been reported to be effective in exon skipping or exon inclusion applications (Fig. 1A). In all cases, these ON analogues mimic RNA in their binding character when forming duplex structures with an RNA target, such that binding is tighter than for DNA analogues. The internucleotide linkage is invariably phosphorothioate (PS), which also allows high protein binding in serum and increases circulation time. All of the ON analogues show high resistance to serum and cellular nucleases. 2′-O-methylphosphorothioate Particularly ubiquitous in exon skipping studies have been 2′-ribose-modified PS ON analogues, the foremost of which is 2′-O-methylphosphorothioate (OMe-PS) (Fig. 1A; Table 1). Exon skipping and functional dystrophin production was demonstrated after intravenous delivery into mdx mice (a standard DMD mouse model) just a few years ago (Lu et al., 2005; Fletcher et al., 2006). OMe-PS quickly became the leading negatively charged ON type in exon skipping, probably because this chemistry is well established for use in vivo and for scale up without onerous license requirements, and OMe-PS ONs appear also to have a relatively good safety profile. Long-term studies have shown that OMe-PS ONs can be administered safely at 200 mg/kg in various DMD mouse models of differing severity for up to 6 months (Tanganyika-de-Winter et al., 2012). An OMe-PS ON (now named Drisapersen) has been taken to clinical trials for exon 51 skipping in DMD patients (Goemans et al., 2011), showing effectiveness in extended treatment in the “6 minute walk test” for ambulatory patients, and phase 3 clinical trial data in Europe are expected imminently. ONs for skipping other exons are also now beginning clinical trials (www.prosensa.eu). OMe-PS ON effectiveness has been reported for other genetic diseases such as exon skipping in the collagen gene in a rat model of dystrophic Epidermolysis Bullosa (Goto et al., 2006) and also for targeting a triplet repeat sequence in the mRNA of the dystrophia myotonica protein kinase (DMPK) in myotonic dystrophy type 1 (Mulders et al., 2009; Gonzalez-Barriga et al., 2013). Exon skipping of ApoB (apolipoprotein B) pre-mRNA by OMe-PS ONs has been shown to reduce LDL cholesterol in ApoB transgenic mice (Disterer et al., 2013). In addition, OMe-PS ONs feature prominently in an approach for use of bifunctional RNAs to target the intronic silencer-N1 to cause exon inclusion, to increase survival motor neuron 1 (SMN1) pre-mRNA levels and to reduce disease severity in an animal model of SMA (Osman et al., 2012). This bifunctional steric block approach had first been proposed some years ago for this disease through cell culture studies (Skordis et al., 2003).