Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.     

Sequence-dependent inhibition of cGAS and TLR9 DNA sensing by 2′-O-methyl gapmer oligonucleotides

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2′OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2′OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2′OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.     

Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides

Currently, gapmer antisense oligonucleotide (ASO) therapeutics are under clinical development for the treatment of various diseases, including previously intractable human disorders; however, they have the potential to induce hepatotoxicity. Although several groups have reported the reduced hepatotoxicity of gapmer ASOs following chemical modifications of sugar residues or internucleotide linkages, only few studies have described nucleobase modifications to reduce hepatotoxicity. In this study, we introduced single or multiple combinations of 17 nucleobase derivatives, including four novel derivatives, into hepatotoxic locked nucleic acid gapmer ASOs and examined their effects on hepatotoxicity. The results demonstrated successful identification of chemical modifications that strongly reduced the hepatotoxicity of gapmer ASOs. This approach expands the ability to design gapmer ASOs with optimal therapeutic profiles.     

Stereo-enriched phosphorothioate oligodeoxynucleotides: synthesis, biophysical and biological properties

Stereo-enriched [Rp] and [Sp]-phosphorothioate oligodeoxynucleotides are synthesized using oxazaphospholidine derivatized monomers. Three different designs of phosphorothioate oligodeoxynucleotides (PS-oligos), (i) stereo-enriched all-[Rp] or all-[Sp] PS-linkages, (ii) stereo-random mixture of PS-linkages, and (iii) segments containing certain number of stereo-enriched [Rp] and [Sp] PS-linkages ([Sp-Rp-Sp] or [Rp-Sp-Rp]), have been studied. Thermal melting studies of these PS-oligos with RNA complementary strands showed that the binding affinities are in the order [Rp] > [Sp-Rp-Sp]-[Rp-Sp-Rp] > stereo-random > [Sp]. Circular dichroism (CD) studies suggest that the stereochemistry of the PS-oligo does not affect the global conformation of the duplex. The in vitro nuclease stability of these PS-oligos is in the order [Sp] > [Sp-Rp-Sp] > stereo-random > [Rp]. The RNase H activation is in the order [Rp] > stereo-random > [Rp-Sp-Rp] > [Sp] > [Sp-Rp-Sp]. Studies in a cancer cell line of PS-oligos targeted to MDM2 mRNA showed that all oligos had similar biological activity under the experimental conditions employed. Protein- and enzyme-binding studies showed insignificant stereo-dependent binding to proteins. The [Sp] and [Sp-Rp-Sp] chimeric and stereo-random PS-oligos that contained a CpG motif showed higher cell proliferation than [Rp] PS-oligo of the same sequence.     

Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction

(Rp)- and (Sp)-5'-O-thymidyl 3'-O-thymidyl [18O]phosphates have been synthesized by reaction of the respective (Sp)- and (Rp)-phosphorothioate precursors with N-bromosuccinimide in dioxane and H218O. Stereochemical analysis of the product derived from the (Rp)-phosphorothioate by digestion with snake venom phosphodiesterase in H217O and examination of the isotopic chirality of the resulting thymidine 5'-[16O,17O,18O]phosphate demonstrate that the replacement reaction has proceeded with inversion of configuration at phosphorus. Inspection of the 31P NMR spectrum of the methyl esters prepared from (Sp)-5'-O-thymidyl 3'-O-thymidyl [18O]phosphate confirms that the replacement reaction has proceeded with very little if any racemization. This spectrum also allows the assignment of the absolute configuration of these methyl triesters. (Rp)-5'-O-Thymidyl 3'-O-thymidyl [18O]phosphate has been used to demonstrate that the stereochemical course of the hydrolytic reaction catalyzed by nuclease P1 from Penicillium citrum proceeds with inversion of configuration at phosphorus and therefore probably does not involve the participation of a covalent enzyme intermediate.     

Progress towards drug discovery for Friedreich’s Ataxia: Identifying synthetic oligonucleotides that more potently activate expression of human frataxin protein

Friedreich’s Ataxia (FRDA) is an incurable genetic disease caused by an expanded trinucleotide AAG repeat within intronic RNA of the frataxin (FXN) gene. We have previously demonstrated that synthetic antisense oligonucleotides or duplex RNAs that are complementary to the expanded repeat can activate expression of FXN and return levels of FXN protein to near normal. The potency of these compounds, however, was too low to encourage vigorous pre-clinical development. We now report testing of “gapmer” oligonucleotides consisting of a central DNA portion flanked by chemically modified RNA that increases binding affinity. We find that gapmer antisense oligonucleotides are several fold more potent activators of FXN expression relative to previously tested compounds. The potency of FXN activation is similar to a potent benchmark gapmer targeting the nuclear noncoding RNA MALAT-1, suggesting that our approach has potential for developing more effective compounds to regulate FXN expression in vivo.     

Influence of a Thiophosphate Linkage on the Duplex Stability - Does Sp Configuration Always Lead to Higher Stability Than Rp?

Abstract

ABSTRACT: In order to design an oligodeoxynucleoside phosphorothioate as an antisense molecule, it is important to establish the structure of the S-oligo with a strong affinity to the target RNA. In these molecules, internucleotide thiophosphate linkages produce diastereomers, the number of which increases in proportion to 2ⁿ (n: number of thiophosphate). To estimate the effect of this linkage on the duplex stability by UV melting curves, oligodeoxynucleotides having a single thiophosphate (referred to Soligo), dGCNsN'CG (s: thiophosphate, N, N′ = A or T), were prepared and their diastereomers isolated by HPLC. As demonstrated previously, the melting temperatures (Tm) for the Sp isomers were higher than those of the Rp when DNA was a target. On the other hand, it was found that for RNA as a target, the Rp isomers of dGCTsTCG and dGCAsTCG had higher stability than the Sp, and that the difference in the Tm values between the diastereomers was smaller than when DNA was a target. With dGCsTsACG, which has two thiophosphates, it was also found that the Tm values decreased with an increase in the number of thiophosphate linkages, and that the difference in Tm between the diastereomers was smaller when RNA was a target. Consequently, in practical clinical applications where RNA is a target, the influence of thiophosphate chirality on the duplex structure is almost negligible and Rp/Sp separation of an S-oligo may be of no major concern.     

β-1,3-Glucan/antisense oligonucleotide complex stabilized with phosphorothioation and its gene suppression

Most of antisense oligonucleotides (ASOs) subjected to current clinical evaluation belong to phosphorothioate (PS) analogues. Although PS has great advantage in DNase resistance, it can induce nonspecific side-effects. Thus it is important to investigate the influence of ASOs with different PS contents. In this paper, we prepared the complex consisting of schizophyllan (SPG) and ASOs attached a dA₄₀ tail with different PS contents to the 3′ end of the ODN, which is introduced to stabilize the complex with SPG. With increase of PS content in the dA₄₀, its complexation ability with SPG was improved and the complex showed high thermal stability. The thermal stability of the fully phosphorothioated ASOs was obtained by only replacing 20% of the oxygen of the phosphodiester moiety. The ability of gene suppression between PS and phosphodiester for antisense sequences was almost the same, indicating that the antisense sequences need not to be PS backbone. These data may provide new insight for the interaction between β-1,3-glucan and DNA and help to deliver therapeutic ODNs.     

Factors Involved in in Vitro Stabilization of Nitrate Reductase from Cotton (Gossypium hirsutum L.) Cotyledons

Experiments were conducted to determine if pretreatment of cotton (Gossypium hirsutum L.) plants resulted in differential in vitro stabilities of nitrate reductase (NR) activity. Although NR activity declines markedly during the second half of the daily light period, in vitro NR stability is not modified by time of harvest. Phenylmethylsulfonylfluoride, iodoacetamide, and N-ethylmaleimide do not influence in vitro NR stability, suggesting that serine or sulfhydryl proteases are not responsible for in vitro lability of NR from cotton cotyledons.

Imposition of water stress or artificial extension of the dark period lead to significant reductions in NR activity, but do not change in vitro NR stability.

Dilution of a crude extract leads to increasing lability of NR; hence the marked instability of NR cannot be attributed to an inactivator which follows simple enzyme kinetics. Since in vitro NR activity is much more stable in presence of both NADH and NO₃⁻, substrate availability must be considered as a possible factor influencing in vivo NR stability.

     

Evaluation of the effect of 2′-O-methyl,fluoro hexitol,bicyclo and Morpholino nucleic acid modifications on potency of GalNAc conjugated antisense oligonucleotides in mice

The potency of antisense oligonucleotide (ASO) drugs has significantly improved in the clinic after exploiting asialoglycoprotein receptor (ASGR) mediated delivery to hepatocytes. To further this technology, we evaluated the structure–activity relationships of oligonucleotide chemistry on in vivo potency of GalNAc-conjugated Gapmer ASOs. GalNAc conjugation improved potency of ASOs containing 2′-O-methyl (2′-O-Me), 3′-fluoro hexitol nucleic acid (FHNA), locked nucleic acid (LNA), and constrained ethyl bicyclo nucleic acid (cEt BNA) 10–20-fold compared to unconjugated ASOs. We further demonstrate that GalNAc conjugation improves activity of 2′-O-(2-methoxyethyl) (2′-O-MOE) and Morpholino ASOs designed to correct splicing of survival motor neuron (SMN2) pre-mRNA in liver after subcutaneous administration. GalNAc modification thus represents a viable strategy for enhancing potency of ASO with diverse nucleic acid modifications and mechanisms of action for targets expressed in hepatocytes.     

Recombinant prosegment peptide acts as a folding catalyst and inhibitor of native pepsin

Porcine pepsin A, a gastric aspartic peptidase, is initially produced as the zymogen pepsinogen that contains an N-terminal, 44 residue prosegment (PS) domain. In the absence of the PS, native pepsin (Np) is irreversibly denatured and when placed under refolding conditions, folds to a thermodynamically stable denatured state. This denatured, refolded pepsin (Rp) state can be converted to Np by the exogenous addition of the PS, which catalyzes the folding of Rp to Np. In order to thoroughly study the mechanism by which the PS catalyzes pepsin folding, a soluble protein expression system was developed to produce recombinant PS peptide in a highly pure form. Using this system, the wild-type and three-mutant PS forms, in which single residue substitutions were made (V4A, R8A and K36A), were expressed and purified. These PS peptides were characterized for their ability to inhibit Np enzymatic activity and to catalyze the folding of Rp to Np. The V4A, R8A and K36A mutant PS peptides were found to have nanomolar inhibition constants, K_i, of 82.4, 58.3 and 95.6 nM, respectively, approximately a two-fold increase from that of the wild-type PS (36.2 nM). All three-mutant PS peptides were found to catalyze Np folding with a rate constant of 0.06 min^? 1, five-fold lower than that of the wild-type. The observation that the mutant PS peptides retained their inhibition and folding-catalyst functionality suggests a high level of resilience to mutations of the pepsin PS.     

Efficacy of an essential oil of Eugenia caryophyllata against Psoroptes cuniculi

The acaricidal activity of Eugenia caryophyllata essential oil was evaluated in vitro and in vivo on Psoroptes cuniculi, a mange mite. In vitro, different concentrations of the oil were tested and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R^®) controls. In vivo, six P. cuniculi infected rabbits were topically treated with the oil diluted at 2.5% and compared with untreated and treated control groups of six rabbits each. In vitro, up to the concentration of 0.10% the oil gave highly significant (P < 0.01) percentages of mite mortality respect to the untreated controls, but only up to 0.16% it showed the same efficacy of Acacerulen R^®. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed respect to the treated control group. The untreated rabbits remained infested.     

TricycloDNA-modified oligo-2'-deoxyribonucleotides reduce scavenger receptor B1 mRNA in hepatic and extra-hepatic tissues--a comparative study of oligonucleotide length, design and chemistry

We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in T(m), cell culture and animal experiments and compare them to their gap-matched 20-mer 2'-O-methoxyethyl (MOE) and 14-mer 2',4'-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar T(m) and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED(50) 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower T(m) and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animals.     

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)_n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.     

The stereochemical course of the first step of pre-mRNA splicing.

We have determined the effects on splicing of sulfur substitution of the non-bridging oxygens in the phosphodiester bond at the 5' splice site of a pre-mRNA intron. Pre-mRNAs containing stereochemically pure Rp and Sp phosphorothioate isomers were produced by ligation of a chemically synthesized modified RNA oligonucleotide to enzymatically synthesized RAs. When these modified pre-mRNA substrates were tested for in vitro splicing activity in a HeLa cell nuclear extract system, the RNA with the Rp diastereomeric phosphorothioate was not spliced while the Sp diastereomeric RNA spliced readily. The sulfur-containing branched trinucleotide was purified from the splicing reaction of the Sp RNA and analyzed by cleavage with a stereospecific nuclease. The results showed that the Sp phosphorothioate was inverted during the splicing reaction to the Rp configuration; a finding previously obtained for a Group I self-splicing RNA. This inversion of configuration is consistent with a transesterification mechanism for pre-mRNA splicing. The lack of splicing of the Rp modified RNA also suggests that the pro-Rp oxygen at the 5' splice site is involved in a critical chemical contact in the splicing mechanism. Additionally, we have found that the HeLa cell RNA debranching enzyme is inactive on branches containing an Rp phosphorothioate.