Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing

Abstract

Oligonucleotides (ONs) have been envisaged for therapeutic applications for more than thirty years. However, their broad use requires overcoming several hurdles such as instability in biological fluids, low cell penetration, limited tissue distribution, and off-target effects. With this aim, many chemical modifications have been introduced into ONs definitively as a means of modifying and better improving their properties as gene silencing agents and some of them have been successful. Moreover, in the search for an alternative way to make efficient ON-based drugs, the general concept of prodrugs was applied to the oligonucleotide field. A prodrug is defined as a compound that undergoes transformations in vivo to yield the parent active drug under different stimuli. The interest in stimuli-responsive ONs for gene silencing functions has been notable in recent years. The ON prodrug strategies usually help to overcome limitations of natural ONs due to their low metabolic stability and poor delivery. Nevertheless, compared to permanent ON modifications, transient modifications in prodrugs offer the opportunity to regulate ON activity as a function of stimuli acting as switches. Generally, the ON prodrug is not active until it is triggered to release an unmodified ON. However, as it will be described in some examples, the opposite effect can be sought. This review examines ON modifications in response to various stimuli. These stimuli may be internal or external to the cell, chemical (glutathione), biochemical (enzymes), or physical (heat, light). For each stimulus, the discussion has been separated into sections corresponding to the site of the modification in the nucleotide: the internucleosidic phosphate, the nucleobase, the sugar or the extremities of ONs. Moreover, the review provides a current and detailed account of stimuli-responsive ONs with the main goal of gene silencing. However, for some stimuli-responsive ONs reported in this review, no application for controlling gene expression has been shown, but a certain potential in this field could be demonstrated. Additionally, other applications in different domains have been mentioned to extend the interest in such molecules.

Keywords: enzymolabile group; light-responsive group; oligonucleotide prodrugs; reduction-responsive; stimuli-responsive nucleic acids; thermolytic prodrugs.

Scheme 1

Demasking under reducing agents of ON prodrugs modified as phosphotriesters with A) benzyl groups [13] and B) a cyclic disulfide trans-5-benzyl-1,2-dithiane-4-yl moiety [14].

Scheme 2

A) Synthesis via phosphoramidite chemistry and B) demasking under the reducing environment of 2’-O-MDTM-modified siRNA prodrugs [17].

Scheme 3

Synthesis via phosphoramidite chemistry of various 2’-O-alkyldithiomethyl (RSSM)-modified RNAs bearing lipophilic or polar groups (R) involving post-elongation conjugation through a thiol disulfide exchange reaction [15].

Scheme 4

A) siRNA conjugates to cholesterol [19] and B) PNA conjugates to a triphenylphosphonium [20] through a disulfide linkage.

Scheme 5

Synthesis via phosphoramidite chemistry and deprotection mediated by nitroreductase/NADH of hypoxia-activated prodrugs of ONs containing A) 5-nitro-2-furylmethyl or 5-nitro-2-thiophenylmethyl [25] and B) 3-(2-nitrophenylpropyl)phosphotriester internucleoside linkages [26].

Scheme 6

Synthesis via phosphoramidite chemistry and conversion mediated by nitroreductase/NADH of hypoxia-activated prodrugs of ONs containing O⁴-(4-nitrobenzyl)thymidine [27].

Scheme 7

Incorporation of O⁶-(4-nitrobenzyl)-2’-deoxyguanosine into an ON prone to form a G-quadruplex structure, preventing it from forming this quadruplex when protected and allowing it under reducing conditions [28].

Scheme 8

Synthesis and mechanism for the demasking of ON prodrugs from A) S-acylthioethyl phosphotriester [29] and B) S-acyloxymethyl phosphotriester [22].

Figure 1

Oligothymidylates bearing A) 2,2-bis(ethoxycarbonyl)-3-(pivaloyloxy)propyl- and B) 2-cyano-2(2-phenylethylaminocarbonyl)-3-(pivaloyloxy)propyl phosphate protecting groups [41].

Figure 2

Oligothymidylates containing esterase and thermo-labile (4-acetylthio-2,2-dimethyl-3-oxobutyl) phosphate protecting groups [42].

Scheme 9

Phosphoramidites and the corresponding RNA prodrugs protected as A) t-Bu-SATE, B) OH-SATE and C) A-SATE phosphotriesters [43].

Scheme 10

Mechanism of the hydrolysis of 2’-O-acyloxymethyl ONs mediated by carboxyesterases [46]. The hydrolysis of the ester functions yields an unstable 2’-hemiacetal, affording the free RNA through the release of formaldehyde.

Scheme 11

Synthesis of partially 2’-O-PivOM-modified RNAs [49] and 2’-O-PiBuOM-modified RNAs [53] using their corresponding phosphoramidites and 2’-O-PrOM phosphoramidites to generate 2’-OH.

Figure 3

A) 2’-O-amino and guanidino-containing acetal ester phosphoramidites and B) 2’-O-(amino acid) acetal ester phosphoramidites reported by Debart [54] and Dahma [55], respectively.

Scheme 12

Prodrugs of tricyclo-ONs functionalized with A) ethyl (tc^ee-T) and B) hexadecyl (tc^hd-T) ester functions at C6 obtained from corresponding thymidine phosphoramidites [56].

Scheme 13

Demasking mechanism of fma thiophosphate triesters in CpG ODN upon heat action [58].

Scheme 14

Thermolytic cleavage of the hydroxy-alkylated thiophosphate and phosphato-/thiophosphato-alkylated thiophosphate protecting groups from thymidine dinucleotides [59].

Scheme 15

Synthesis via phosphoramidite chemistry and thermolytic cleavage of alkylated (diisopropyl, diethyl, morpholino) phosphoramidothioylbutyl internucleoside linkages [61].

Scheme 16

Synthesis of thermosensitive prodrugs of ODNs containing fma thiophosphate triesters combined to positively charged 3-(N,N-dimethylamino)propyl phosphotriesters internucleoside linkages to improve cellular uptake [62].

Scheme 17

Caging of deoxycytidine in methylphosphonate ONs by using the thermolabile phenylsulfonylcarbamoyl protecting group introduced through reaction with phenylsulfonyl isocyanate [65].

Figure 4

Biotinylated 1-(5-(aminomethyl)-2-nitrophenyl)ethyl phosphoramidite used to cage the 5’-end of a siRNA during its synthesis on solid support using phosphoramidite chemistry [73].

Scheme 18

Introduction and cleavage of 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) [74] and cyclododecyl-DMNPE (CD-DMNPE) [76] groups in the terminal 3’ and 5’-phosphate of an RNA through reaction with a diazo reagent.

Scheme 19

Post-synthetic introduction of a thioether-enol phosphodiester (TEEP) linkage into a DNAzyme by the selective reaction of a phosphorothioate linkage with 2-bromo-4’-hydroxyacetophenone followed by photodecaging, leading to a phosphodiester internucleoside linkage [77].

Scheme 20

A) NPP dT and dG phosphoramidites [–92] and B) NPOM U and G phosphoramidites [83] used to introduce photocaged nucleobases into siRNAs C) close to the argonaute cleavage site to prevent siRNA cleavage [81,83] and D) in the seed region to prevent mRNA recognition by the RISC complex [83].

Scheme 21

Introduction of the photocaged 3-NPOM nucleobase into phosphorothioate antisense and morpholino antisense to inhibit RNA translation though mRNA degradation by RNase H [84] or steric blocking [85].

Scheme 22

Control of the activity of an antisense ODN using a photocaged hairpin [82]. Formation of the hairpin suppresses hybridization of the antisense ODN with mRNA, which could be translated.

Scheme 23

Control of alternative splicing using phosphorothioate (PS) 2’-OMe-photocaged ONs resulting from the incorporation of 3-NPOM 2’-OMe uridine phosphoramidite [86]. Photoirradiation activates the ODN, inducing a correct splicing.

Scheme 24

A) Light activation of a photocaged DNAzyme incorporating 3-NPOM thymidine in its catalytic site [87]; B) light deactivation of a photocaged DNAzyme by formation of an inactive hairpin [82].

Scheme 25

Incorporation of 3-(6-nitropiperonyloxymethyl) (NPOM) thymidine and 4-nitropiperonylethyl (NPE) deoxycytidine phosphoramidites into TFOs and light inhibition and light activation of gene transcription using caged TFOs and caged hairpin TFOs, respectively [88].

Scheme 26

Synthesis of a photocaged DNA decoy from a 3-NPOM thymidine phosphoramidite and release of the NPOM protecting group under photolysis, allowing the decoy to organize into a hairpin that can bind to the NF-κB transcription factor [89].

Scheme 27

Synthesis of a caged DNA decoy hairpin containing a 7-nitroindole nucleotide and release of the modified nucleobase under photolysis, leading to an abasic lactone-containing ON that cannot form a hairpin and associate with NF-κB [90].

Figure 5

Caged-2’-adenosines used by MacMillan et al [–94] (X = O) and Piccirilli et al [95] (X = S) to study RNA mechanisms.

Scheme 28

Synthesis of circular ODNs containing a photolabile linker as described by Tang et al. [101,104].

Scheme 29

Control of RNA digestion with RNase H using light activation of a photocaged hairpin [97].

Scheme 30

Photocontrol of RNA degradation using caged circular antisense ODNs containing a photoresponsive linker [101].

Scheme 31

Control of RNA translation using an “RNA bandage” consisting of two short 2’-OMe ONs linked together with a photosensitive linker [100].

Scheme 32

Control of alternative splicing using photocaged ONs resulting from the incorporation of an o-nitrobenzyl responsive moiety as its phosphoramidite [86]. Photoirradiation deactivates the ODN, inducing incorrect splicing.

Scheme 33

A) Light deactivation of a photocaged DNAzyme incorporating one photocleavable spacer in its catalytic site and another in the recognition site; B) light activation of a circular photocaged DNAzyme formed through the hybridization and ligation of the DNAzyme with a complementary strand [103].

Scheme 34

Solid-phase synthesis of a caged vit E-siRNA conjugate and its release upon UV irradiation [106].

Scheme 35

Synthesis of a siRNA conjugated to a nanoparticle (NP) via a cyclooctene heterolinker from a siRNA-NH₂ and an NP-NH₂ [107]. The conjugate does not induce gene silencing until tetrazine triggers siRNA release.