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Antisense technology: A review

Open AccessPublished:February 15, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100416
      Antisense technology is beginning to deliver on the broad promise of the technology. Ten RNA-targeted drugs including eight single-strand antisense drugs (ASOs) and two double-strand ASOs (siRNAs) have now been approved for commercial use, and the ASOs in phase 2/3 trials are innovative, delivered by multiple routes of administration and focused on both rare and common diseases. In fact, two ASOs are used in cardiovascular outcome studies and several others in very large trials. Interest in the technology continues to grow, and the field has been subject to a significant number of reviews. In this review, we focus on the molecular events that result in the effects observed and use recent clinical results involving several different ASOs to exemplify specific molecular mechanisms and specific issues. We conclude with the prospective on the technology.

      Keywords

      Abbreviations:

      ds (double-stranded), FCS (familial chylomicronemia), GalNAc (N-acetyl galactosamine), GFR (glomerular filtration rate), PD (pharmacodynamics), PK (pharmacokinetic), RTD (RNA-targeted drug), SGLT2 (sodium-glucose cotransmitter 2), SMN1 (survival motor neuron), ss ASO (single-strand antisense oligonucleotide), TIE (translation inhibitory element)
      Recent progress and the performance of RNA-targeted drugs (RTDs) in well-controlled clinical trials argue that RTD discovery technology is beginning to deliver the potential value that encouraged investment beginning 3 decades ago. To date, ten RTDs have been approved for commercial use (Table 1), including eight single-strand antisense oligonucleotide (ss ASO) drugs (ASOs) and two double-stranded (ds) antisense drugs that are referred to as siRNAs (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Seth P.P.
      • Vickers T.A.
      • Liang X.H.
      The interaction of phosphorothioate-containing RNA targeted drugs with proteins is a critical determinant of the therapeutic effects of these agents.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ). Nusinersen, a PS ASO that corrects the splicing of the SMN2 pre-mRNA to treat spinal muscular atrophy (SMA) is the first “blockbuster” RTD (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ). Though all of the approved RTDs are designed to treat patients with rare diseases, Table 1 shows that while the rare disease pipeline is robust, there are numerous RTDs in advanced clinical trials that are focused on diseases with very high incidences. In fact, Pelacarsen, formerly AKCEA-APO(a)-LRx and ION-TTR- LRx, is enrolling patients into cardiovascular outcome studies and in advanced phase 2 studies, there are PS ASOs such as IONIS-FXI-LRx, IONIS APOCIII-LRx, Vupanorsen (formerly AKCEA-ANGPTL3-LRx), and a number of ASOs designed to treat high-incidence neurological diseases including treatments for Alzheimer’s and Parkinson’s diseases (Table 1) (
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ). Table 1 also shows that ss PS ASOs can be administered by multiple routes for both systemic and local therapeutic effects. The versatility of the technology is further demonstrated by the fact that post-RNA-binding mechanisms of action include both ASOs that cause RNA reduction via RNase H1 and those that increase protein production by correcting RNA splicing defects. Preclinical data suggest that in the coming years, a range of new post-RNA-binding mechanisms will be used to bring clinical benefit (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Liang X.H.
      • Nichols J.G.
      • Hsu C.W.
      • Vickers T.A.
      • Crooke S.T.
      mRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway.
      ,
      • Liang X.H.
      • Sun H.
      • Shen W.
      • Wang S.
      • Yao J.
      • Migawa M.T.
      • Bui H.H.
      • Damle S.S.
      • Riney S.
      • Graham M.J.
      • Crooke R.M.
      • Crooke S.T.
      Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.
      ,
      • Liang X.-h.
      • Shen W.
      • Sun H.
      • Migawa M.T.
      • Vickers T.A.
      • Crooke S.T.
      Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames.
      ). That ASOs in the clinic are being used to treat diseases caused in full or partly by toxic RNAs, as well as more traditional protein-caused diseases argue that more targets and opportunities will emerge as the roles of RNAs are better understood. Finally, several chemical classes and the critical importance of advances in medicinal chemistry are apparent. The significance of advances in oligonucleotide medicinal chemistry and in ASO designs is best shown by comparing the performances of phosphorothioate (PS) oligodeoxynucleotides (first generation) to those PS ASOs containing 2ʹ methoxyethyl (2ʹ-MOE) substitutions (second generation) to PS ASOs containing 2ʹ constrained ethyl (2ʹcEt) modifications (generation 2.5) to 2ʹ-MOE and 2ʹcEt PS ASOs conjugated with N-acetyl galactosamine (GalNAc) (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ).
      Table 1Clinical development activities of antisense medicines
      A - approved RNA targeted drugs
      DrugIndicationTarget RNAMechanismChemistryYear approved
      FomivirsenCytomegalovirus retinitisHCMV UL122RNase H1PS, DNA1998
      MipomersenHomozygous familial hypercholesterolemiaAPOBRNase H1PS, MOE2013
      NusinersenSpinal muscular atrophySMN2Splicing, intron 7PS, MOE2016
      EteplirsenDuchenne muscular dystrophyDMDSplicing, exon 51PMO2016
      InotersenHereditary transthyretin- mediated amyloidosisTTRRNase H1PS, MOE2018
      VolanesorsenFamilial chylomicronemia syndromeApoC-IIIRNase H1PS, MOE2019
      PatisiranHereditary transthyretin- mediated amyloidosisTTRAgo2PO-siRNA

      Cationic lipid formulation
      2018
      GolodirsenDuchenne muscular dystrophyDMDSplicing, exon 53PMO2019
      GivosiranAcute hepatic porphyriaALAS1Ago2Ome/F- siRNA, GalNAc2019
      ViltolarsenDuchenne muscular dystrophyDMDSplicing, exon 53PMO2020
      B. ASO drugs under development
      Chemistry
      All drugs are modified with PS linkages, except for the PMOs. All 2’-MOE chemistries include 2’-deoxy sugar residues to support Rnase H1 activity, unless specified as fully modified. Abbreviations are provided by column heading. Chemistry: 2’-H, 2’-deoxy; 2’-F, 2’-fluoro; 2’-Ome, 2’-methoxy; 2’- MOE, 2’-O-methoxy ethyl; cET, (S)-constrained ethyl; ENA, 2’-O,4’-C-ethylene-bridged nucleic acid; GalNAc, triantennary N-acetylgalactosamine; LNA, locked nucleic acid; PMO, phosphorodiamidate morpholino oligomer; PO, phosphodiester linkage; PS, phosphorothioate linkage; SNA, spherical nucleic acid nanoparticle. Drugs: 2.5, generation 2.5 ASO drugs containing cEt modification; L, ligand conjugate. Target/Organ: AGT, angiotensinogen; ANGPTL3, angiopoietin like 3; Apo(a), apolipoprotein A1; ApoB-100, apolipoprotein B-100; ApoC-III, apolipoprotein C-III; AR, androgen receptor; C9orf72, chromosome 9 open reading frame 72; CD49d, integrin subunit alpha 4; CEP290, centrosomal protein 290; CLU, clusterin; CMV IE2, cytomegalovirus immediate early gene 2; COL7A1, collagen type VII alpha 1 chain; DGAT2, diacylglycerol O-acyltransferase 2; DMD, dystrophin; DMPK, DM1 protein kinase; FGFR4, fibroblast growth factor receptor 4; GCCR, glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1); GCGR, glucagon receptor; GHR, growth hormone receptor; GRB2, growth factor receptor bound protein 2; HBV, hepatitis B virus; HCV, hepatitis C virus; HSP27, heat shock protein 27; HTT, huntingtin; ICAM1, intercellular adhesion molecule 1; IL17RA, interleukin 17 receptor alpha; KLKB1, kallikrein B1; LRRK2, leucine rich repeat kinase 2; MAPT (TAU), microtubule-associated protein tau; mHTT, mutant huntingtin; miR, microRNA; PCSK9, proprotein convertase subtilisin/kexin type 9; PTP1B, protein tyrosine phosphatase, nonreceptor type 1; RHO, Rhodopsin; SCNN1A (EnaC), sodium channel epithelial 1 alpha subunit; SGLT2, sodium/glucose cotransporter 2 (solute carrier family 5 member 2); SMAD7, SMAD family member 7; SMN2, survival of motor neuron 2; SNCA, alpha-synuclein; SOD1, superoxide dismutase 1; STAT3, signal transducer and activator of transcription 3; TGF-β2, transforming growth factor beta 2; TNFα, tumor necrosis factor alpha; TTR, transthyretin; USH2A, usherin. Dose/Route: IM, intramuscular; IT, intrathecal; ITM, intratumoral; IV, intravenous; IVT, intravitreal; SC, subcutaneous. Indication: ALS, amyotrophic lateral sclerosis; AML, acute myeloid leukemia; ATTR, transthyretin amyloidosis; CML, chronic myelogenous leukemia; CMV, cytomegalovirus; CVD, cardiovascular disease; DMD, Duchenne muscular dystrophy; FCS, familial chylomicronemia syndrome; HAE, hereditary angioedema; HBV, hepatitis B virus; HCV, hepatitis C virus; HoFH, homozygous familial hypercholesterolemia; MD, myotonic dystrophy; MS, multiple sclerosis; NASH, nonalcoholic steatohepatitis; NSCLC, non-small-cell lung cancer; SMA, spinal muscular atrophy; T2D, type 2 diabetes. Key Observations: AIDS, acquired immune deficiency syndrome; ALT, alanine aminotransferase; CTC, circulating tumor cells; FDA, Food and Drug Administration; HbA1c, hemoglobin A1c; IL6, interleukin 6; LDL, low-density lipoprotein; Lp(a), lipoprotein (a); PSA, prostate antigen. References: NCT, national clinical trial (registry, clinicaltrials.gov).
      DrugTarget | OrganDose | RouteIndicationKey observationsReferences
      Phase 3
       2ʹ-MOETofersen (BIIB067/ISIS 666853)SOD1, CNSALS (Ionis/Biogen)Phase 2 findings of dose-dependent reduction in SOD1 CSF concentration; slowing decline in clinical function, respiratory function, and muscle strength, versus placebo; well tolerated with multiple dose administrations at doses of 20–100 mgNCT02623699 (
      • Miller T.
      • Cudkowicz M.
      • Shaw P.J.
      • Andersen P.M.
      • Atassi N.
      • Bucelli R.C.
      • Genge A.
      • Glass J.
      • Ladha S.
      • Ludolph A.L.
      • Maragakis N.J.
      • McDermott C.J.
      • Pestronk A.
      • Ravits J.
      • Salachas F.
      • et al.
      Phase 1-2 trial of antisense oligonucleotide tofersen for SOD1 ALS.
      )
       2ʹ-MOETominersen (RG6042/ISIS 443139)HTT, CNS10−120 mg once every 4 weeks, ITHuntington’s Disease (Ionis/Roche)Phase ½a findings of dose-dependent reduction of mutant huntingtin CSF concentration; well tolerated with multiple dose administrations at monthly doses of 10–120 mgNCT03761849 NCT02519036 (
      • Tabrizi S.J.
      • Leavitt B.R.
      • Landwehrmeyer G.B.
      • Wild E.J.
      • Saft C.
      • Barker R.A.
      • Blair N.F.
      • Craufurd D.
      • Priller J.
      • Rickards H.
      • Rosser A.
      • Kordasiewicz H.B.
      • Czech C.
      • Swayze E.E.
      • Norris D.A.
      • et al.
      Targeting huntingtin expression in patients with Huntington's disease.
      )
       undisclosedSepofarsen (QR-110)LCA10, p.Cys998XITVLeber’s Congenital Amaurosis Type 10 (ProQR)Phase ½ findings included improvement in visual acuityNCT03913143
       2ʹ-MOE, GalNAcPelacarsen (TQJ230/AKCEA- APO(a)-LRx)Apo(a), Liver80 mg once monthly, SCCVD (Ionis/Akcea/Novartis)Phase 2 findings of dose-dependent reduction in serum lipoprotein (a) levels; no difference compared with placebo-treated patients in platelet counts, liver and renal function testsNCT04023552 NCT03070782 (
      • Tsimikas S.
      • Karwatowska-Prokopczuk E.
      • Gouni-Berthold I.
      • Tardif J.C.
      • Baum S.J.
      • Steinhagen-Thiessen E.
      • Shapiro M.D.
      • Stroes E.S.
      • Moriarty P.M.
      • Nordestgaard B.G.
      • Xia S.
      • Guerriero J.
      • Viney N.J.
      • O'Dea L.
      • Witztum J.L.
      • et al.
      Lipoprotein(a) reduction in persons with cardiovascular disease.
      )
       2ʹ-MOE, GalNAcAKCEA- TTR-LRx (ION 682884)TTR, Liver45 mg once monthly, SCATTR (Ionis/Akcea)Phase 1 findings of dose-dependent reduction in plasma TTR; well tolerated with multiple dose administrations; no clinically relevant effect on platelet counts, or liver and renal function testsNCT04136184 NCT04136171 NCT03728634 (
      • Viney N.J.
      • Tai L.
      • Jung S.
      • Yu R.Z.
      • Guthrie S.
      • Baker B.F.
      • Geary R.S.
      • CSchneider E.
      • Guo S.
      • Monie B.P.
      Phase 1 investigation of a ligand- conjugated antisense oligonucleotide with increased potency for the treatment of transthyretin amyloidosis.
      )
       PMOCasimersen (SRP-4045)Dystrophin Exon 45, Muscle30 mg/kg once weekly, IVDMD (Sarepta)Statistically significant increase from baseline in dystrophin protein (versus placebo) at week 48 interim end point Study ongoing (double-blind, placebo- controlled to 96 weeks)NCT02500381 (
      • Therapeutics Sarepta
      Sarepta Therapeutics Announces Positive Expression Results from the Casimersen (SRP-4045) Arm of the ESSENCE Study.
      )
       2ʹ-HAlicaforsenICAM-1, Colon240 mg once daily, EnemaChronic Pouchitis and Ulcerative Colitis (Ionis)Phase 2 findings included reduction in Pouchitis Disease Activity Index and endoscopy subscoreNCT02525523 (
      • Miner Jr., P.B.
      • Wedel M.K.
      • Xia S.
      • Baker B.F.
      Safety and efficacy of two dose formulations of alicaforsen enema compared with mesalazine enema for treatment of mild to moderate left-sided ulcerative colitis: A randomized, double-blind, active-controlled trial.
      ,
      • Miner P.
      • Wedel M.
      • Bane B.
      • Bradley J.
      An enema formulation of alicaforsen, an antisense inhibitor of intercellular adhesion molecule-1, in the treatment of chronic, unremitting pouchitis.
      )
      Phase 2
       2ʹ-MOEBIIB080 (IONIS- MAPTRx)TAU, CNSOnce monthly, ITAlzheimer’s Disease, FTD (Ionis/Biogen)NCT03186989
       2ʹ-MOEIONIS-FXIRx/BAY 2306001Factor XI, Liver100–300 mg once weekly, SCClotting Disorders (Ionis/Bayer)Phase 2 findings included reduction of Factor XI protein, and reduction of thrombotic events without increase in bleeding.NCT02553889 NCT01713361 (
      • Buller H.R.
      • Bethune C.
      • Bhanot S.
      • Gailani D.
      • Monia B.P.
      • Raskob G.E.
      • Segers A.
      • Verhamme P.
      • Weitz J.I.
      • Investigators F.-A.T.
      Factor XI antisense oligonucleotide for prevention of venous thrombosis.
      )
       2ʹ-MOEIONIS- GCGRRxGCGR, Liver50−200 mg once weekly, SCT2D (Ionis/Suzhou-Ribo)Phase 2 findings included attenuation of glucagon-induced increase in blood glucose levels, dose-dependent reduction of HbA1c, no cases of severe or symptomatic hypoglycemia, dose- dependent increase in liver transaminase levels consistent with pharmacology, and no increase in hepatic glycogen contentNCT01885260 NCT02583919 NCT02824003 (
      • Morgan E.S.
      • Tai L.J.
      • Pham N.C.
      • Overman J.K.
      • Watts L.M.
      • Smith A.
      • Jung S.W.
      • Gajdosik M.
      • Krssak M.
      • Krebs M.
      • Geary R.S.
      • Baker B.F.
      • Bhanot S.
      Antisense inhibition of glucagon receptor by IONIS-GCGRRx improves type 2 diabetes without increase in hepatic glycogen content in patients with type 2 diabetes on stable metformin therapy.
      )
       2ʹ-MOEIONIS- DGAT2RxDGAT2, Liver250 mg once weekly, SCNASH (Ionis)Phase 2 findings included significant absolute reduction in liver fat, versus placebo, and 50% of patients treated had at least a 30% relative reduction in liver fat; no changes in liver or renal function, no cases of thrombocytopeniaNCT03334214 (
      • Loomba R.
      • Morgan E.
      • Watts L.
      • Xia S.
      • Hannan L.A.
      • Geary R.S.
      • Baker B.F.
      • Bhanot S.
      Novel antisense inhibition of diacylglycerol O-acyltransferase 2 for treatment of non-alcoholic fatty liver disease: A multicentre, double-blind, randomised, placebo-controlled phase 2 trial.
      )
       2ʹ-MOEApatorsenHSP27, Tumor Cells200−1000 mg once weekly, IVCancer (Ionis/OncoGenex)Phase 1 findings included decrease in tumor markers and decline in CTCsNCT01454089 (
      • Chi K.N.
      • Yu E.Y.
      • Jacobs C.
      • Bazov J.
      • Kollmannsberger C.
      • Higano C.S.
      • Mukherjee S.D.
      • Gleave M.E.
      • Stewart P.S.
      • Hotte S.J.
      A phase I dose- escalation study of apatorsen (OGX-427), an antisense inhibitor targeting heat shock protein 27 (Hsp27), in patients with castration-resistant prostate cancer and other advanced cancers.
      )
       2ʹ-MOEATL1102CD49d, Immune CellsDMD: 25 mg weekly, SC MS: 200 mg twice weekly, SCDMD, MS (Ionis/ATL)Phase 2 findings in nonambulatory DMD patients included positive effects on modulating CD49d+ T cells in blood Phase 2 findings in MS patients included reduction in new active lesions, and moderate thrombocytopeniaACTRN1261800 0970246 (
      • Limmroth V.
      • Barkhof F.
      • Desem N.
      • Diamond M.P.
      • Tachas G.
      CD49d antisense drug ATL1102 reduces disease activity in patients with relapsing-remitting MS.
      )
       2ʹ-MOEAtesidorsen/ATL1103GHR, Liver200 mg once or twice weekly, SCAcromegaly (Ionis/ATL)Phase 2 findings included significant reduction in IGF-1 in patients with acromegaly who received ATL1103 200 mg twice weekly, versus once weeklyACTRN1261500 0289516 (
      • Trainer P.J.
      • Newell-Price J.D.C.
      • Ayuk J.
      • Aylwin S.J.B.
      • Rees A.
      • Drake W.
      • Chanson P.
      • Brue T.
      • Webb S.M.
      • Fajardo C.
      • Aller J.
      • McCormack A.I.
      • Torpy D.J.
      • Tachas G.
      • Atley L.
      • et al.
      A randomised, open- label, parallel group phase 2 study of antisense oligonucleotide therapy in acromegaly.
      )
       2ʹ-MOEIONIS- HBVRxHBV surface Ag, Liver150–300 mg once weekly, SCHBV, chronic atypical (Ionis/GSK)Phase 2 findings included dose dependent reductions of HbsAg and HBV DNA, and an acceptable safety profile to proceed to longer treatment durationsNCT02981602 (
      • Yuen M.F.
      • Jang J.-W.
      • Jang J.-H.
      • Yoon J.-H.
      • Kweon Y.-O.
      • Park S.-J.
      • Bennett C.F.
      • Kwoh T.J.
      Phase 2a, randomized, double-blind, placebo controlled study of an antisense inhibitor (ISIS 505358) in treatment-naïve chronic hepatitis B (CHB) patients: Safety and antiviral efficacy.
      )
       2ʹ-Ome

      Stereo-pure PS
      WVE-120101mHTT (rs362307), CNSITHuntington’s Disease (Wave)In progressNCT03225833
       2ʹ-Ome

      Stereo-pure PS
      WVE-120102mHTT (rs362331), CNS2–32 mg, ITHuntington’s Disease (Wave)In progressNCT03225846
       cEtDYN101DNM2, MTM11.5–9.0 mg/kg, IMCentronuclear Myopathy (Ionis/Dynacure)In progressNCT04033159
       cETIONIS- ENAC-2.5RxENAC, LungInhaled/NebulizedCystic Fibrosis (Ionis)In progressNCT03647228
       cETAZD9150/IONIS- STAT3-2.5RxSTAT3, Cancer and Stromal Cells2−4 mg/kg once weekly, IVCancer (Ionis/Astrazeneca)Phase 1b findings included (1) reduction of STAT3, (2) reduction in serum IL6, and (3) reduction in tumor burden.NCT02549651 NCT01563302 (
      • Reilley M.J.
      • McCoon P.
      • Cook C.
      • Lyne P.
      • Kurzrock R.
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      • Woessner R.
      • Younes A.
      • Nemunaitis J.
      • Fowler N.
      • Curran M.
      • Liu Q.
      • Zhou T.
      • Schmidt J.
      • Jo M.
      • et al.
      STAT3 antisense oligonucleotide AZD9150 in a subset of patients with heavily pretreated lymphoma: Results of a phase 1b trial.
      ,
      • Hong D.
      • Kurzrock R.
      • Kim Y.
      • Woessner R.
      • Younes A.
      • Nemunaitis J.
      • Fowler N.
      • Zhou T.
      • Schmidt J.
      • Jo M.
      • Lee S.J.
      • Yamashita M.
      • Hughes S.G.
      • Fayad L.
      • Piha- Paul S.
      • et al.
      AZD9150, a next- generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung cancer.
      )
       cETAZD5312/IONIS-AR- 2.5RxAR, Cancer Cells150−1150 mg once weekly, IVProstate cancer (Ionis/Suzhou- Ribo)Phase 1 findings included declines in PSA and circulating tumor cells in some patients.NCT03300505 NCT02144051 (
      • Chowdhury S.
      • Burris H.A.
      • Patel M.
      • Infante J.R.
      • Jones S.F.
      • Voskoboynik M.
      • Parry K.
      • Elvin P.
      • Coleman T.
      • Gardner H.
      • Lyne P.D.
      • Arkenau H.T.
      A phase I dose escalation, safety and pharmacokinetic (PK) study of AZD5312 (IONIS- ARRx), a first-in-class generation 2.5 antisense oligonucleotide targeting the androgen receptor (AR).
      )
       LNACobomarsen (MRG-106)miR-155, Cancer Cells75−900 mg once weekly, ITM/SC/IVHematological malignancies (miRagen)Phase 1 findings included improvements in cutaneous lesions, and transcriptional changes consistent with target activityNCT03713320 NCT02580552 (
      • Querfeld C.
      • Foss F.M.
      • Pinter-Brown L.C.
      • Porus P.
      • William B.M.
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      • HGuitart J.
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      • DeSimone J.
      • Seto A.G.
      • Pestano L.A.
      • Jackson A.L.
      • Williams P.J.
      • et al.
      Phase 1 study of the safety and efficacy of MRG-106, a synthetic inhibitor of microRNA-155, in CTCL patients.
      )
       LNACivi 007PCSK9, LiverSCCVD (Civi)In progressNCT04164888 NCT03427710
       PO, 2ʹ-Ome, ENADS-5141bDystrophin Exon 45, Muscle0.1−6.0 mg/kg once weekly, SCDMD (Daiichi)In progressNCT02667483
       PMO PeptideSRP-5051Dystrophin Exon 51, MuscleMultiple ascending dose, IVDMD (Sarepta)In progressNCT04004065 NCT03375255
       2ʹ-MOE, GalNAcVupanorsen (AKCEA- ANGPTL3-LRx/ION 702803)ANGPTL3, Liver40–80 mg total monthly dose, SCDyslipidemias (Ionis/Akcea/Pfizer)Phase 2 findings in patients with HTG, T2D and NAFLD included dose-dependent reductions in ANGPTL3, TGs, ApoC-III, VLDL and non-HDL cholesterol, and total cholesterol with no reductions in liver fat or HbA1c; favorable safety and tolerability profileNCT03371355 NCT02709850 (
      • Graham M.J.
      • Lee R.G.
      • Brandt T.A.
      • Tai L.J.
      • Fu W.
      • Peralta R.
      • Yu R.
      • Hurh E.
      • Paz E.
      • McEvoy B.W.
      • Baker B.F.
      • Pham N.C.
      • Digenio A.
      • Hughes S.G.
      • Geary R.S.
      • et al.
      Cardiovascular and metabolic effects of ANGPTL3 antisense oligonucleotides.
      ,
      • Gaudet D.
      • Karwatowska-Prokopczuk E.
      • Baum S.J.
      • Hurh E.
      • Kingsbury J.
      • Bartlett V.J.
      • Figueroa A.L.
      • Piscitelli P.
      • Singleton W.
      • Witztum J.L.
      • Geary R.S.
      • Tsimikas S.
      • St L.O.D.L.
      Vupanorsen, an N-acetyl galactosamine-conjugated antisense drug to ANGPTL3 mRNA, lowers triglycerides and atherogenic lipoproteins in patients with diabetes, hepatic steatosis, and hypertriglyceridaemia.
      )
       2ʹ-MOE, GalNAcAKCEA- APOCIII-LRxApoC-III, Liver10–50 mg total monthly dose, SCCVD (Ionis/Akcea)Phase 2 findings in patients with HTG and CVD, or high-risk of CVD, included >90% patients at 50 mg monthly dose achieved TG ≤ 150 mg/dl compared to 5% placebo, significant reduction in multiple risk factors, and no safety signals, including those related to platelet counts, liver or renal functionNCT03385239 NCT02900027 (
      • Alexander V.J.
      • Xia S.
      • Hurh E.
      • Hughes S.G.
      • O'Dea L.
      • Geary R.S.
      • Witztum J.L.
      • Tsimikas S.
      N-acetyl galactosamine-conjugated antisense drug to APOC3 mRNA, triglycerides and atherogenic lipoprotein levels.
      ,
      Akcea TherapeuticsIonis Pharmaceuticals
      Akcea and Ionis Report Positive Topline Phase 2 Results of AKCEA-APOCIII-L Rx.
      )
       2ʹ-MOE, GalNAcIONIS-AGT-LRxAGT, LiverOnce weekly, SCTreatment-resistant Hypertension (Ionis)In progressNCT04083222 NCT03714776 NCT03101878
       2ʹ-MOE, GalNAcGSK3389404/IONIS- HBV-LRxHBV surface Ag, Liver30−120 mg single dose/once weekly, SCChronic HBV (Ionis/GSK)In progressNCT03020745 (
      • Yuen M.
      • Heo J.
      • Kumada H.
      • Suzuki F.
      • Suzuki Y.
      • Xie Q.
      • Jia J.
      • HKarino Y.
      • Hou J.
      • Chayama K.
      • Imamura M.
      • Iao-Tao J.Y.
      • Lim S.G.
      • Tanaka Y.
      • Xie W.
      • et al.
      Results After 12 Weeks Treatment of Multiple Doses of GSK3389404 in Chronic Hepatitis B (CHB) Subjects on Stable Nucleos(t)ide Therapy in a Phase 2a Double-Blind, Placebo- Controlled Study.
      )
       2ʹ-MOE, GalNAcIONIS-FB-LRxFactor B, Liver10−40 mg once every 2 weeks, SCPrimary IgA Nephropathy Ocular Disease (Ionis/Roche)Phase 1 findings included (1) dose- dependent reduction in factor B levels accompanied by similar reduction in factor B function and complement split factor Bb, and (2) no drug-related adverse eventsNCT04014335 NCT03815825 ACTRN1261600 0335493 (
      • Grossman T.R.
      • Carrer M.
      • Shen L.
      • Johnson R.B.
      • Hettrick L.A.
      • Henry S.P.
      • Monia B.P.
      • McCaleb M.L.
      Reduction in ocular complement factor B protein in mice and monkeys by systemic administration of factor B antisense oligonucleotide.
      )
       2ʹ-MOE, GalNAcIONIS-PKK-LRxKallikrein B1, Liver20–80 mg, once monthly, SCHAE (Ionis)Phase 1 findings included dose- dependent reduction of plasma prekallikrein levels with target reduction maintained during dosing intervals as predicted by PK propertiesNCT04307381 NCT03263507 (
      • Crooke S.T.
      • Baker B.F.
      • Xia S.
      • Yu R.Z.
      • Viney N.J.
      • Wang Y.
      • Tsimikas S.
      • Geary R.S.
      Integrated assessment of the clinical performance of GalNAc3- conjugated 2'-O-methoxyethyl chimeric antisense oligonucleotides: I. Human volunteer experience.
      ,
      • Cohn D.M.
      • Viney N.J.
      • Fijen L.M.
      • Schneider E.
      • Alexander V.J.
      • Xia S.
      • Kaeser G.E.
      • Nanavati C.
      • Baker B.F.
      • Geary R.S.
      • Levi M.
      • Meijers J.C.M.
      • Stroes E.S.G.
      Antisense inhibition of Prekallikrein to control hereditary angioedema.
      )
       2ʹ-MOE, GalNAcIONIS-GHR-LRxGHR, LiverMonthly, SCAcromegaly (Ionis)In progressNCT03548415
       2ʹ-MOE, GalNAcIONIS-FXI-LRxFXI, LiverMonthly, SCClotting Disorders (Ionis/Bayer)In progressNCT03582462
       PO, 2ʹ-H LiposomePrexigebersenGRB2, Tumor cellsTwice weekly, IVAML, CML, solid tumors (Bio-Path)In progressNCT04196257 NCT02923986 NCT02781883
       UndisclosedQR-1123 (ION357)RHO, P23H mutation, EyeITVAutosomal Dominant Retinitis Pigmentosa (Ionis/ProQR)In progressNCT04123626
       UndisclosedQR-421aUSH2A, exon 13 mutation, Eye50–200 μg, ITVRetinitis pigmentosa (ProQR)In progressNCT03780257
       UndisclosedQR-313COL7A1, exon 73 mutation, SkinOnce daily, topical creamRecessive dystrophic epidermolysis bullosa (ProQR/Wings)In progressNCT03605069
       UndisclosedRG-012miR-21, Kidney110−220 mg once weekly, SCAlport Syndrome (Regulus/Genzyme)In progressNCT02855268
      Phase 1
       2ʹ-MOEBIIB078 (IONIS- C9Rx)C9orf72, CNSMultiple ascending doses, ITALS (Ionis/Biogen)In progressNCT04288856 NCT03626012
       2ʹ-MOEBIIB094 (ION859)LRRK2, CNSSingle and multiple ascending doses, ITParkinson’s Disease (Ionis/Biogen)In progressNCT03976349
       2ʹ-MOEBIIB101 (ION464)SNCA, CNSITMultiple System Atrophy (Ionis/Biogen)NCT04165486
       LNARG6127Host targetSCHBV (Roche)NCT03762681
       LNAISTH0036TGF-β2, Eye6.75−225 μg single dose, IVTGlaucoma (Isarna)Phase 1 findings included (1) dose–response trend observed in postoperative introcular pressure, and (2) no adverse eventsNCT02406833 (
      • Pfeiffer N.
      • Voykov B.
      • Renieri G.
      • Bell K.
      • Richter P.
      • Weigel M.
      • Thieme H.
      • Wilhelm B.
      • Lorenz K.
      • Feindor M.
      • Wosikowski K.
      • Janicot M.
      • Packert D.
      • Rommich R.
      • Mala C.
      • et al.
      First-in-human phase I study of ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-beta2), in subjects with open-angle glaucoma undergoing glaucoma filtration surgery.
      )
       SNA Lipid nanoparticleXCUR17IL17RA, SkinDaily, Topical gelPsoriasis (Exicure)Phase 1 finding included decrease in the levels of psoriasis and inflammation markers downstream of target, with a significant reduction in keratin 16 expression and clinical improvement in epidermal thickness(
      • Giljohann D.
      Clinical results from XCUR17, a topically applied antisense spherical nucleic acid in patients with psoriasis.
      )
       UndisclosedJNJ- 64991524UndisclosedOralUndisclosed (Janssen)NCT03346122
      Nonactive
       2ʹ-MOEIONIS- PTP1BRxPTP1B, Liver200 mg once weekly, SCT2D (Ionis)Phase 2 findings included (1) reduction of HbA1c, (2) improved leptin and adiponectin levels, and (3) decreased body weight.NCT00455598 (
      • Digenio A.
      • Pham N.C.
      • Watts L.M.
      • Morgan E.S.
      • Jung S.W.
      • Baker B.F.
      • Geary R.S.
      • Bhanot S.
      Antisense inhibition of protein tyrosine phosphatase 1B with IONIS-PTP-1BRx improves insulin sensitivity and reduces weight in overweight patients with type 2 diabetes.
      )
       2ʹ-MOEIONIS- GCCRRxGCCR, Liver60−420 mg once weekly, SCT2D (Ionis)Phase 1 findings included (1) improvement in lipid profile, and (2) attenuation of dexamethasone-induced hepatic insulin resistance.NCT01968265 (
      • Bhanot S.
      • Morgan E.
      • Bethune C.
      • Larouche R.
      • S X.
      • Geary R.
      ISIS- GCCRRX, a novel glucocorticoid (GC) receptor antisense drug reduces cholesterol and triglycerides and attenuates dexamethasone induced hepatic insulin resistance without systemic GC antagonism in normal subjects.
      )
       2ʹ-MOEIONIS- FGFR4RxFGFR4, Liver100−200 mg once weekly, SCObesity (Ionis)NCT02463240
       LNAMiravirsenmiR-122, Liver3−7 mg/kg once weekly, SCHCV (Santaris/Roche)Phase 2 findings included inhibition of miR-122 functionNCT01200420 (
      • Janssen H.L.
      • Reesink H.W.
      • Lawitz E.J.
      • Zeuzem S.
      • Rodriguez-Torres M.
      • Patel K.
      • van der Meer A.J.
      • Patick A.K.
      • Chen A.
      • Zhou Y.
      • Persson R.
      • King B.D.
      • Kauppinen S.
      • Levin A.A.
      • Hodges M.R.
      Treatment of HCV infection by targeting microRNA.
      ,
      • van der Ree M.H.
      • van der Meer A.J.
      • de Bruijne J.
      • Maan R.
      • van Vliet A.
      • Welzel T.M.
      • Zeuzem S.
      • Lawitz E.J.
      • Rodriguez-Torres M.
      • Kupcova V.
      • Wiercinska- Drapalo A.
      • Hodges M.R.
      • Janssen H.L.
      • Reesink H.W.
      Long-term safety and efficacy of microRNA-targeted therapy in chronic hepatitis C patients.
      )
      Discontinued
       Stereo-pure PS 2ʹ-F, 2ʹ-OmeSuvodirsenDystrophin Exon 51, Muscle3.5–5.0 mg/kg weekly, IV infusionDMD (Wave)Failure to demonstrate efficacy in multidose phase 1 open-label extensionNCT03907072 NCT03508947
       2ʹ-MOEIONIS- PKKRxKallikrein B1, Liver200 mg once weekly, SCHAE

      Chronic Migraine (Ionis)
      Replaced with GalNAc conjugateNCT03108469 (
      • Cohn D.M.
      • Viney N.J.
      • Fijen L.M.
      • Schneider E.
      • Alexander V.J.
      • Xia S.
      • Kaeser G.E.
      • Nanavati C.
      • Baker B.F.
      • Geary R.S.
      • Levi M.
      • Meijers J.C.M.
      • Stroes E.S.G.
      Antisense inhibition of Prekallikrein to control hereditary angioedema.
      ,
      • Ferrone J.D.
      • Bhattacharjee G.
      • Revenko A.S.
      • Zanardi T.A.
      • Warren M.S.
      • Derosier F.J.
      • Viney N.J.
      • Pham N.C.
      • Kaeser G.E.
      • Baker B.F.
      • Schneider E.
      • Hughes S.G.
      • Monia B.P.
      • MacLeod A.R.
      IONIS-PKKRx a novel antisense inhibitor of prekallikrein and bradykinin production.
      )
       2ʹ-HMongersenSMAD7, Intestine160 mg once daily, OralCrohn’s Disease (Nogra Pharma/Celgene)Phase 3 failed to demonstrate benefit in patients with active Crohn’s Disease with clinical remission (CD Activity Index score <150) attained in 22.8% of patients on GED-0301 versus 25% on placebo (p = 0.6210). Adverse eventsNCT02596893 NCT02601300 (
      • Sands B.E.
      • Feagan B.G.
      • Sandborn W.J.
      • Schreiber S.
      • Peyrin-Biroulet L.
      • Frederic Colombel J.
      • Rossiter G.
      • Usiskin K.
      • Ather S.
      • Zhan X.
      • D'Haens G.
      Mongersen (GED-0301) for active Crohn's disease: Results of a phase 3 study.
      ,
      • Monteleone G.
      • Pallone F.
      Mongersen, an oral SMAD7 antisense oligonucleotide, and Crohn's disease.
      )
       2ʹ-OmeDrisapersenDystrophin Exon 51, Muscle6 mg/kg once weekly, SCDMD (Prosensa/BioMarin)Rejected by FDA(
      • Goemans N.M.
      • Tulinius M.
      • van den Hauwe M.
      • Kroksmark A.K.
      • Buyse G.
      • Wilson R.J.
      • van Deutekom J.C.
      • de Kimpe S.J.
      • Lourbakos A.
      • Campion G.
      Long-term efficacy, safety, and pharmacokinetics of drisapersen in Duchenne muscular dystrophy: Results from an open-label extension study.
      ,
      • Voit T.
      • Topaloglu H.
      • Straub V.
      • Muntoni F.
      • Deconinck N.
      • Campion G.
      • De Kimpe S.J.
      • Eagle M.
      • Guglieri M.
      • Hood S.
      • Liefaard L.
      • Lourbakos A.
      • Morgan A.
      • Nakielny J.
      • Quarcoo N.
      • et al.
      Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): An exploratory, randomised, placebo- controlled phase 2 study.
      ,
      • McCarty T.
      • Charlesworth D.
      BioMarin Announces Withdrawal of Market Authorization Application for Kyndrisa™ (Drisapersen) in Europe.
      )
       2ʹ-MOECustirsenCLU, Tumor Cells640 mg once weekly, IVProstate Cancer and NSCLC (Ionis/OncoGenex)Failure to meet primary endpoints in phase 3 trialsNCT01578655 NCT01630733 (
      • Saad F.
      • Hotte S.
      • North S.
      • Eigl B.
      • Chi K.
      • Czaykowski P.
      • Wood L.
      • Pollak M.
      • Berry S.
      • Lattouf J.B.
      • Mukherjee S.D.
      • Gleave M.
      • Winquist E.
      • Canadian Uro- Oncology G.
      Randomized phase II trial of Custirsen (OGX-011) in combination with docetaxel or mitoxantrone as second-line therapy in patients with metastatic castrate-resistant prostate cancer progressing after first-line docetaxel: CUOG trial P-06c.
      ,
      OncoGenex Pharmaceuticals, Inc.
      OncoGenex Announces Results from the Phase 3 AFFINITY Trial of Custirsen in Men with Metastatic Castrate-Resistant Prostate Cancer.
      )
       2ʹ-MOEIONIS- APO(a)RxApo(a), Liver300 mg once weekly, SCCVD (Ionis)Replaced with GalNAc conjugate(
      • Tsimikas S.
      • Viney N.J.
      • Hughes S.G.
      • Singleton W.
      • Graham M.J.
      • Baker B.F.
      • Burkey J.L.
      • Yang Q.
      • Marcovina S.M.
      • Geary R.S.
      • Crooke R.M.
      • Witztum J.L.
      Antisense therapy targeting apolipoprotein(a): A randomised, double-blind, placebo-controlled phase 1 study.
      )
       2ʹ-MOEISIS 388626SGLT2, Kidney50−200 mg once weekly, SCT2D (Ionis)Availability of small-molecule inhibitors of SGLT2(
      • van Meer L.
      • Moerland M.
      • van Dongen M.
      • Goulouze B.
      • de Kam M.
      • Klaassen E.
      • Cohen A.
      • Burggraaf J.
      Renal effects of antisense-mediated inhibition of SGLT2.
      )
       2ʹ-MOEISIS 333611SOD1, CNS0.15−3.0 mg single dose, ITFamilial ALS (Ionis)Replaced by more potent compound(
      • Miller T.M.
      • Pestronk A.
      • David W.
      • Rothstein J.
      • Simpson E.
      • Appel S.H.
      • Andres P.L.
      • Mahoney K.
      • Allred P.
      • Alexander K.
      • Ostrow L.W.
      • Schoenfeld D.
      • Macklin E.A.
      • Norris D.A.
      • Manousakis G.
      • et al.
      An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: A phase 1, randomised, first-in-man study.
      )
       2ʹ-MOEISIS 104838TNFα, Immune Cells0.1−6 mg/kg IV, 200 mg once weekly, SCInflammatory Disease (Ionis)Inadequate activity(
      • Sewell K.L.
      • Geary R.S.
      • Baker B.F.
      • Glover J.M.
      • Mant T.G.
      • Yu R.Z.
      • Tami J.A.
      • Dorr F.A.
      Phase I trial of ISIS 104838, a 2'-methoxyethyl modified antisense oligonucleotide targeting tumor necrosis factor-alpha.
      )
       2ʹ-MOEISIS 113715PTP1B, Liver100−600 mg once weekly, SCT2D (Ionis)Replaced by more potent compound
       cET, 2ʹ-MOEIONIS- DMPK2.5RxDMPK, Muscle100−600 mg once weekly, SCMD type 1 (Ionis)Inadequate activityNCT02312011 (
      • Mignon L.
      • Norris D.
      • Bishop K.
      • Derosier F.
      • LANE R.
      • Bennett F.
      ISIS- DMPKRx in healthy volunteers: A placebo-controlled, randomized, single ascending- dose phase 1 study (P3.166).
      )
       LNAEZN-4176AR, Cancer Cells0.5−10 mg/kg once weekly, IVProstate CancerALT elevationsNCT01337518 (
      • Bianchini D.
      • Omlin A.
      • Pezaro C.
      • Lorente D.
      • Ferraldeschi R.
      • Mukherji D.
      • Crespo M.
      • Figueiredo I.
      • Miranda S.
      • Riisnaes R.
      • Zivi A.
      • Buchbinder A.
      • Rathkopf D.E.
      • Attard G.
      • Scher H.I.
      • et al.
      First-in-human phase I study of EZN-4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor mRNA in patients with castration-resistant prostate cancer.
      )
       Undisclosed, GalNAcAZD4076/RG-125miR-103/107, LiverSCDiabetic NASH (Regulus/Astra Zeneca)Failed in developmentNCT02826525
       Undisclosed, GalNAcRG-101miR-122, LiverSCHCV (Regulus)Cases of hyperbilirubinemiaEudraCT 2016–002069–77 (
      Regulus Therapeutics
      Regulus Announces Pipeline Updates and Advancements.
      )
      a All drugs are modified with PS linkages, except for the PMOs. All 2’-MOE chemistries include 2’-deoxy sugar residues to support Rnase H1 activity, unless specified as fully modified. Abbreviations are provided by column heading. Chemistry: 2’-H, 2’-deoxy; 2’-F, 2’-fluoro; 2’-Ome, 2’-methoxy; 2’- MOE, 2’-O-methoxy ethyl; cET, (S)-constrained ethyl; ENA, 2’-O,4’-C-ethylene-bridged nucleic acid; GalNAc, triantennary N-acetylgalactosamine; LNA, locked nucleic acid; PMO, phosphorodiamidate morpholino oligomer; PO, phosphodiester linkage; PS, phosphorothioate linkage; SNA, spherical nucleic acid nanoparticle. Drugs: 2.5, generation 2.5 ASO drugs containing cEt modification; L, ligand conjugate. Target/Organ: AGT, angiotensinogen; ANGPTL3, angiopoietin like 3; Apo(a), apolipoprotein A1; ApoB-100, apolipoprotein B-100; ApoC-III, apolipoprotein C-III; AR, androgen receptor; C9orf72, chromosome 9 open reading frame 72; CD49d, integrin subunit alpha 4; CEP290, centrosomal protein 290; CLU, clusterin; CMV IE2, cytomegalovirus immediate early gene 2; COL7A1, collagen type VII alpha 1 chain; DGAT2, diacylglycerol O-acyltransferase 2; DMD, dystrophin; DMPK, DM1 protein kinase; FGFR4, fibroblast growth factor receptor 4; GCCR, glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1); GCGR, glucagon receptor; GHR, growth hormone receptor; GRB2, growth factor receptor bound protein 2; HBV, hepatitis B virus; HCV, hepatitis C virus; HSP27, heat shock protein 27; HTT, huntingtin; ICAM1, intercellular adhesion molecule 1; IL17RA, interleukin 17 receptor alpha; KLKB1, kallikrein B1; LRRK2, leucine rich repeat kinase 2; MAPT (TAU), microtubule-associated protein tau; mHTT, mutant huntingtin; miR, microRNA; PCSK9, proprotein convertase subtilisin/kexin type 9; PTP1B, protein tyrosine phosphatase, nonreceptor type 1; RHO, Rhodopsin; SCNN1A (EnaC), sodium channel epithelial 1 alpha subunit; SGLT2, sodium/glucose cotransporter 2 (solute carrier family 5 member 2); SMAD7, SMAD family member 7; SMN2, survival of motor neuron 2; SNCA, alpha-synuclein; SOD1, superoxide dismutase 1; STAT3, signal transducer and activator of transcription 3; TGF-β2, transforming growth factor beta 2; TNFα, tumor necrosis factor alpha; TTR, transthyretin; USH2A, usherin. Dose/Route: IM, intramuscular; IT, intrathecal; ITM, intratumoral; IV, intravenous; IVT, intravitreal; SC, subcutaneous. Indication: ALS, amyotrophic lateral sclerosis; AML, acute myeloid leukemia; ATTR, transthyretin amyloidosis; CML, chronic myelogenous leukemia; CMV, cytomegalovirus; CVD, cardiovascular disease; DMD, Duchenne muscular dystrophy; FCS, familial chylomicronemia syndrome; HAE, hereditary angioedema; HBV, hepatitis B virus; HCV, hepatitis C virus; HoFH, homozygous familial hypercholesterolemia; MD, myotonic dystrophy; MS, multiple sclerosis; NASH, nonalcoholic steatohepatitis; NSCLC, non-small-cell lung cancer; SMA, spinal muscular atrophy; T2D, type 2 diabetes. Key Observations: AIDS, acquired immune deficiency syndrome; ALT, alanine aminotransferase; CTC, circulating tumor cells; FDA, Food and Drug Administration; HbA1c, hemoglobin A1c; IL6, interleukin 6; LDL, low-density lipoprotein; Lp(a), lipoprotein (a); PSA, prostate antigen. References: NCT, national clinical trial (registry, clinicaltrials.gov).
      Though traditional drug discovery modalities have advanced incrementally, to a very large extent, these technologies have remained static, dependent on cumbersome, costly, and time-consuming screening processes and are highly inefficient. Rather remarkably, given how much the technology has already advanced and of vital importance is the fact that advances in antisense technology are continuing. Deeper understanding of the molecular mechanisms responsible for the pharmacokinetic (PK) behaviors of PS ASOs in animals and cells, the molecular mechanisms of pharmacodynamic (PD) effects of PS ASOs and the toxicities are yielding ever better performing PS ASOs. Most exciting is that antisense medicinal chemistry, which for most of the 3 decades of research on antisense technology focused on enhancing ASO interactions with target RNAs, now adds a major focus on PS ASO–protein interactions and targeted delivery to specific tissues such as what has been achieved with GalNAc conjugation (
      • Setten R.L.
      • Rossi J.J.
      • Han S.P.
      The current state and future directions of RNAi-based therapeutics.
      ,
      • Nair J.K.
      • Attarwala H.
      • Sehgal A.
      • Wang Q.
      • Aluri K.
      • Zhang X.
      • Gao M.
      • Liu J.
      • Indrakanti R.
      • Schofield S.
      • Kretschmer P.
      • Brown C.R.
      • Gupta S.
      • Willoughby J.L.S.
      • Boshar J.A.
      • et al.
      Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.
      ). The progress in the technology and its current status have been the subject of several recent reviews (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Seth P.P.
      • Vickers T.A.
      • Liang X.H.
      The interaction of phosphorothioate-containing RNA targeted drugs with proteins is a critical determinant of the therapeutic effects of these agents.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ).
      Nevertheless, whether the technology has the potential to be truly broadly enabling remains to be answered in full. In this review, we will first address this question by describing the known properties of PS ASOs that lead us to believe that the technology has the potential to be even more broadly enabling than small-molecule drug discovery. This will be followed by a more detailed discussion than has been provided in other reviews of the theoretical framework that was the basis on which progress was made and on which the future of the technology will be written. Then, the current understanding of the molecular mechanisms responsible for the effects of ASOs that have been observed will be comprehensively discussed. Finally, we will get a glimpse of the new advances and insights that are defining the future.

      Why antisense technology?

      The idea of designing oligonucleotides to bind to specific sequences in target RNAs via Watson–Crick hydrogen bonding and the term “antisense” were introduced in 1978 (
      • Zamecnik P.C.
      • Stephenson M.L.
      Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide.
      ). As proposed, the idea was quite simple and the word antisense was nonspecific. The authors were agnostic as to the post-RNA-binding mechanisms that would ensue to alter the behavior and performance of a target RNA, the structure of drug administered (single or double strand) or the chemical modifications that might be required to introduce acceptable properties for therapeutic administration. “Antisense” included an oligonucleotide of any structure or chemistry designed to bind target RNA via Watson–Crick hybridization. In practice, antisense is used to refer to ss ASOs, and siRNA refers to ds ASOs that act via AGO2 to cause degradation of target RNAs (for review see (
      • Sigova A.
      • Zamore O.D.
      Small RNA silencing pathways.
      ,
      • Chernikov I.V.
      • Vlassov V.V.
      • Chernolovskaya E.L.
      Current development of siRNA bioconjugates: From research to the clinic.
      )).
      Not until the late 1980s, however, did any meaningful effort to convert the concept to reality begin when several companies were founded to pursue the concept. The delay is easily explained because most considered the concept impossible to reduce to practice. And for good reasons. It was impossible to cost-effectively synthesize even gram quantities of an oligonucleotide. It was clear that unmodified oligonucleotides could not be effective therapeutic agents because they are rapidly degraded by nucleases present in all in vitro and in vivo biological systems. Nor had any oligonucleotide medicinal chemistry had been performed, and it was unclear what the scope of the effort might be. Because of their size and negative charge, theoretical concepts of the time argued that these agents could not enter cells and without entering cells, ASOs could not function via an antisense mechanism. Moreover, experience with polynucleotides such as poly(I): poly(C) suggested that oligonucleotides would be unacceptably toxic (
      • Field A.K.
      • Young C.W.
      • Krakoff I.H.
      • Tytell A.A.
      • Lampson G.P.
      • Nemes M.M.
      • Hilleman M.R.
      Induction of interferon in human subjects by poly I:C.
      ). Given the daunting challenges, why invest the decades, careers, and billions of dollars required to learn whether the concept could be reduced to practice? The simple answer was the limitations of traditional approaches to drug discovery.

      Limitations of traditional drug discovery approaches

      The intellectual basis of the drug discovery industry was defined around the end of the 19th century independently by Langley, Erlich, and Bernard (
      • Maehle A.-H.
      • Prüll C.-R.
      • Halliwell R.F.
      The emergence of the drug receptor theory.
      ). As the theories of the time were inadequate, all were wrestling with the same question: how could one explain the potency of certain toxins such as curare and arsenicals? Ultimately, the term “receptive substance” was coined to suggest a specific interaction between these toxins and some substance in the body that bound with higher affinity than other sites (for review, see (
      • Maehle A.H.
      “Receptive substances”: John Newport Langley (1852-1925) and his path to a receptor theory of drug action.
      )). It is fascinating that an entire industry and thousands of products, to say nothing of the benefits of the products, were created prior to the 1980s when the concept was validated with the purification of the nicotinic receptor (for review, see (
      • Changeux J.P.
      The nicotinic acetylcholine receptor: The founding father of the pentameric ligand-gated ion channel superfamily.
      )) and the cloning of the first receptors (for review, see (
      • Finlay D.B.
      • Duffull S.B.
      • Glass M.
      100 years of modelling ligand-receptor binding and response: A focus on GPCRs.
      )). Though the term receptor or receptive substance was entirely generic, in practice a receptor has been considered a protein and, even today, despite progress in RNA-targeted drug discovery, many pharmacologists assume that receptors are proteins.
      Contemporaneously, chemical manufacturers, seeking new applications for their products, found that small molecules such as acetylsalicylic acid, or aspirin, could be effective therapeutics. Thus, traditional small-molecule drug discovery was birthed. The effects of various small-molecule agents were rationalized in the context of receptor theory, leading to the evolution of small-molecule drug discovery. Over time, the so-called 500 Da rule, which suggests that ideal therapeutic agents should be no greater than 500 Da, was established with a focus on designing small molecules designed to bind to specific proteins, either cell surface proteins coupled to signal transduction processes, i.e., receptors, or enzymes involved in cellular processes such as intermediary metabolism (
      • Abou-Gharbia M.
      Discovery of innovative small molecule therapeutics.
      ). Though crude plant extracts had been used for thousands of years as therapeutics, the idea of purifying the active component from biological sources for therapeutic purposes developed later. Arguably the first example of a “purified” natural product was insulin as replacement therapy for type 1 diabetes (
      • Vecchio I.
      • Tornali C.
      • Bragazzi N.L.
      • Martini M.
      The discovery of insulin: An important milestone in the history of medicine.
      ), and protein replacement therapies have added meaningful value for a relatively limited number of diseases. It was not until the use of penicillin during the Second World War that large-scale fermentation and purification of natural products to be used as antiinfectives were proven feasible. The virtual eradication of bacterial infections as a cause of significant mortality in the developed world remains perhaps the greatest achievement of the drug discovery industry (
      • Jackson N.
      • Czaplewski L.
      • Piddock L.J.V.
      Discovery and development of new antibacterial drugs: Learning from experience?.
      ). Interestingly, natural products are typically chemically more diverse and higher molecular weight than the “500 Da rule.” A single novel drug discovery technology was reasonably advanced at the time, but still not fully proven and of course that was monoclonal antibody technology. Over the last 3 decades, this technology has advanced and has proven to be an important dug discovery platform, used relatively broadly, but certainly not as broadly enabling as small-molecule drug discovery (
      • Goding J.W.
      Introduction to monoclonal antibodies.
      ).
      Clearly, the great strength of small-molecule drug discovery is that there are small-molecule drugs that are pharmacologically active after all routes of administration, in all the organs and most cells in the body. However, the limitations are well known and those limits have shaped the industry: inadequate selectivity, numerous “undruggable” targets, inefficient drug discovery and development, the inability to learn general principles from past experience. (The phrase “change a methyl, change the drug” is sadly true.)
      The major reason to invest in the creation and advancement of antisense technology was that it had the potential to address all the limitations of small-molecule drug discovery. Evolutionary processes chose pattern recognition in nucleic acids polymers, as the means to store, retrieve, and use critical genetic information because of the specificity afforded by the genetic code. Since ASOs were to be designed to use the same code, the rules that govern binding of ASOs to RNA were well understood and could be directly applied to the drug discovery process. Moreover, the basic rules governing the specificity of binding to nucleic acid sequences were simple enough to calculate and then prove experimentally that 16 to 20 nucleotides were optimal (
      • Monia B.P.
      • Johnston J.F.
      • Geiger T.
      • Muller M.
      • Fabbro D.
      Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-Raf kinase.
      ). Consequently, in principle, drug discovery should be significantly more efficient and the drugs should be more specific than small molecules. It was further hoped that essentially all targets should be druggable since the biosynthesis of proteins depends on an RNA that should be targetable. The efficiency of drug development might also be greatly enhanced because all ASOs within the same chemical class differ only in sequence, thus should share many properties in common and the failure rate of drugs in development should be reduced because the lessons learned with an ASO representative of a specific chemical class could be applied (
      • Crooke S.T.
      • Vickers T.A.
      • Lima W.F.
      • Wu H.-J.
      Mechanisms of antisense drug action, an introduction.
      ). These were powerful incentives to invest, and the advantages have been to a large extent realized. The evidence supporting these conclusions and the questions that remain will be discussed. As several thorough reviews of the technology have recently been published that reviewed recent clinical results in detail (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Seth P.P.
      • Vickers T.A.
      • Liang X.H.
      The interaction of phosphorothioate-containing RNA targeted drugs with proteins is a critical determinant of the therapeutic effects of these agents.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ), we will not reiterate the summary of recent clinical results. Rather, we will discuss the results of selected clinical studies to exemplify novel specific mechanisms and the impacts of those advances.

      History

      Prerequisite advances

      Without the evolution in understanding the RNA world (Fig. 1), antisense technology would not be possible. Because of continuing advances in understanding the structures and functions of RNAs, new opportunities for antisense technology continue to be identified. In fact, a simple and correct way to think of antisense drugs is that they alter the intermediary metabolism of RNAs. ASOs can be designed to alter the anabolism of precursor RNAs to mature RNAs, including, pre-mRNAs (
      • Sazani P.
      • Graziewicz M.A.
      • Kole R.
      Splice switching oligonucleotides as potential therapeutics.
      ,
      • Hodges D.
      • Crooke S.T.
      Inhibition of splicing of wild-type and mutated luciferase-adenovirus pre-mRNAs by antisense oligonucleotides.
      ), pre-rRNA (
      • Pollak A.J.
      • Liang X.-H.
      • Crooke S.T.
      Gapmer ASOs targeting 5S rRNA can reduce mature 5S rRNA by two mechanisms.
      ), long noncoding RNAs (
      • Shen W.
      • Liang X.H.
      • Sun H.
      • De Hoyos C.L.
      • Crooke S.T.
      Depletion of NEAT1 lncRNA attenuates nucleolar stress by releasing sequestered P54nrb and PSF to facilitate c-Myc translation.
      ), snRNAs and snoRNAs (
      • Liang X.H.
      • Vickers T.A.
      • Guo S.
      • Crooke S.T.
      Efficient and specific knockdown of small non-coding RNAs in mammalian cells and in mice.
      ), antisense transcripts (
      • Crooke S.T.
      • Vickers T.A.
      • Lima W.F.
      • Wu H.-J.
      Mechanisms of antisense drug action, an introduction.
      ), and toxic RNAs such as those containing triplet repeats (
      • Swinnen B.
      • Robberecht W.
      • Van Den Bosch L.
      RNA toxicity in non- coding repeat expansion disorders.
      ). ASOs can also be designed to alter the catabolism of precursor and mature RNAs via several mechanisms (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Liang X.H.
      • Nichols J.G.
      • Hsu C.W.
      • Vickers T.A.
      • Crooke S.T.
      mRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway.
      ) and to alter translation (
      • Liang X.H.
      • Sun H.
      • Shen W.
      • Wang S.
      • Yao J.
      • Migawa M.T.
      • Bui H.H.
      • Damle S.S.
      • Riney S.
      • Graham M.J.
      • Crooke R.M.
      • Crooke S.T.
      Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.
      ,
      • Liang X.-h.
      • Shen W.
      • Sun H.
      • Migawa M.T.
      • Vickers T.A.
      • Crooke S.T.
      Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Lima W.F.
      • Wu H.-J.
      Mechanisms of antisense drug action, an introduction.
      ).
      Figure thumbnail gr1
      Figure 1Antisense technology can modulate gene expression through different mechanisms. A, commonly used antisense mechanisms to degrade target RNAs, including RNase H1-dependent and RISC-dependent mechanisms. B, various types of RNAs. In addition to the three traditional types of RNAs (rRNAs, tRNAs, and mRNAs) that are directly involved in protein synthesis, many types of small noncoding RNAs (<200 nt) have been identified to participate in various biological processes: this includes snRNAs in pre-mRNA splicing, snoRNAs in rRNA processing and modification, miRNAs in translation modulation and mRNA stability, piRNAs in RNA silencing and epigenetics, and other small ncRNAs (e.g., RNase P and MRP RNA) in tRNA and rRNA maturation. Moreover, a large number of long ncRNAs (>200 nt) have also been identified in recent years to play important roles in multiple biological processes, from chromatin remodeling (e.g., Xist, HOTAIR), transcription (e.g., asRNA, eRNA), and RNA processing (e.g., NATS) in the nucleus to translation (e.g., as-Uchl1) and protein modification (e.g., NKILA) in the cytoplasm. In addition, circular RNAs have also been identified that may modulate gene expression by acting as sponges for mRNAs or RNA-binding proteins. C, antisense oligonucleotides can modulate gene expression through additional mechanisms, including downregulation by induced NMD through altering splicing (upper left), inhibit translation initiation by binding to cap region or triggering mRNA no-go decay by binding to coding region of mRNAs (upper right panels). In addition, antisense oligonucleotides can also upregulate gene expression by altering splicing to skip PTC containing exons (lower left panel) or by inhibiting EJC binding to inhibit NMD (lower middle panel) or by enhancing translation through masking translation inhibitory elements, including uORF, TIE, miRNA-binding sites or even miRNAs (lower right panel). The circles of different colors indicate proteins. CDS, coding region sequence; EJC, exon–exon junction complex; NMD, non-sense-mediated decay; PTC, premature termination codon; RISC, RNA-induced silencing complex; TIE, translation inhibit element; uORF, upstream open reading frame. The red bars indicate antisense oligonucleotides or miRNAs.
      Similarly, the sequences of target RNAs must be available to support the design of ASOs. Thus, advances in DNA sequencing that ultimately led to the sequencing of the human and other genomes (
      • McCombie W.R.
      • McPherson J.D.
      • Mardis E.R.
      Next-generation sequencing technologies.
      ) are a prerequisite for antisense drug discovery. Not only are modern genomic sequencing and transcriptomic methods essential for target selection, but they also support approaches to prove mechanisms of action and the selectivity of RNA target reduction (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ).

      Intellectual framework

      An essential first step in the creation of antisense technology was the elaboration of an intellectual framework in which ASOs could be considered. Though chemically modified oligonucleotides had never been considered as possible therapeutic agents prior to the advent of antisense technology and specific sequences in targeted RNAs, it made sense to rationalize behaviors of ASOs in the context of traditional receptor theory (
      • Crooke S.T.
      Antisense drug technology: Principles, strategies, and applications.
      ). As trivial as this may seem, framing the questions that needed to be answered in this context has been critical.

      Broad-based medicinal chemistry efforts

      ASOs are chemically modified oligonucleotides. This is because phosphodiester oligonucleotides are rapidly degraded by exo- and endonucleases and lack the necessary affinity for receptor sequences in target RNAs. Though a great deal of medicinal chemistry had been performed on nucleosides, no focused effort to create the medicinal chemistry of oligonucleotides had been performed in the early years. Thus, a broad-based program that modified essentially every position in an oligonucleotide, except sites required for Watson–Crick hydrogen bonding, was developed and many thousands of modifications have synthesized and tested in vitro and in vivo. These modifications include those involving the nucleobase, the sugar, and the phosphate. Additionally, numerous groups of diverse structures have been conjugated at the 5ʹand 3ʹ termini and various positions on the nucleobase and the 2ʹ position of the sugar (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ). However, based on first principles, the major focus was on the 2ʹ position of the sugar and the phosphate, and indeed, the major advances in performance derived from modifications at these sites. Since ds ASOs or siRNAs are comprised of the same building blocks, the medicinal chemistry created to modify ASOs served as the basic chemical tool kit for siRNAs as well (
      • Setten R.L.
      • Rossi J.J.
      • Han S.P.
      The current state and future directions of RNAi-based therapeutics.
      ,
      • Sigova A.
      • Zamore O.D.
      Small RNA silencing pathways.
      ,
      • Chernikov I.V.
      • Vlassov V.V.
      • Chernolovskaya E.L.
      Current development of siRNA bioconjugates: From research to the clinic.
      ,
      • Manoharan M.
      • Rajeev K.G.
      Utilizing chemistry to harness RNA interference pathways for therapeutics: Chemically modified siRNAs and antagomirs.
      ,
      • Tang X.
      • Zhang S.
      • Fu R.
      • Zhang L.
      • Huang K.
      • Peng H.
      • Dai L.
      • Chen Q.
      Therapeutic prospects of mRNA-based gene therapy for glioblastoma.
      ). Because these advances have been stochastic, they have been dubbed second-generation and generation 2.5 chemistries (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ).
      More recently with the success of GalNAc conjugation in targeted delivery of both ss PS ASOs and ds PS containing siRNAs (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Setten R.L.
      • Rossi J.J.
      • Han S.P.
      The current state and future directions of RNAi-based therapeutics.
      ,
      • Tang X.
      • Zhang S.
      • Fu R.
      • Zhang L.
      • Huang K.
      • Peng H.
      • Dai L.
      • Chen Q.
      Therapeutic prospects of mRNA-based gene therapy for glioblastoma.
      ), focus has shifted to targeted delivery to other tissues. Additionally, because of breakthroughs in understanding the molecular mechanisms by which PS ASOs induce toxicities, site-specific modifications have evolved (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Shen W.
      • De Hoyos C.L.
      • Migawa M.T.
      • Vickers T.A.
      • Sun H.
      • Low A.
      • Bell 3rd, T.A.
      • Rahdar M.
      • Mukhopadhyay S.
      • Hart C.E.
      • Bell M.
      • Riney S.
      • Murray S.F.
      • Greenlee S.
      • Crooke R.M.
      • et al.
      Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.
      ).

      The molecular mechanisms by which ASOs produce their effects

      The major advances here are focused primarily on PS ASOs and have included insights into the mechanisms of cellular uptake and distribution, pharmacodynamics, and cellular and organ toxicity, and these in turn have resulted in new approaches to enhance the performance of PS ASOs (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ,
      • Crooke S.T.
      Molecular mechanisms of antisense oligonucleotides.
      ,
      • Crooke S.T.
      • Wang S.
      • Vickers T.A.
      • Shen W.
      • Liang X.H.
      Cellular uptake and trafficking of antisense oligonucleotides.
      ).

      Process, analytical and manufacturing chemistries

      In 1989, the synthesis of even gram quantities of PS ASOs was impossible and prohibitively costly. Similarly, analytical methods to determine the purity of PS ASOs were primitive as were methods to measure PS ASOs and metabolites in biological systems. Advances in these areas were critical to the development first of PS ASOs for rare diseases, then as potency increased, the synthesis of the ton quantities of PS ASOs necessary for very large indications. Similarly, analytical methods have improved dramatically in precision and sensitivity (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Capaldi D.C.
      • Scozzari A.N.
      Manufacturing and analytical processes for 2O-(2-methoxy-ethyl)-modified oligonucleotides.
      ).
      In fact, considered from a process chemical perspective, scale-up was comparatively simple as there were no new chemistries that needed to be invented, no exotherms, or high-pressure reactions, and PS ASOs are quite stable when stored as dry powders. Today, at ton scale, cost of goods is sufficiently low to support commercialization for very large indications and even oral administration.

      Structural classes of RTDs

      Depending on the length, ss PS (or PO)-modified ASOs range from approximately 6.5 to 7.5 kDa. Single-stranded molecules behave as highly flexible rods and can adopt a wide range of conformations in solution. Waters of hydration and counterions are present at each PS (or PO) linkage (
      • White A.P.
      • Reeves K.K.
      • Snyder E.
      • Farrell J.
      • Powell J.W.
      • Mohan V.
      • Griffey R.H.
      Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides.
      ,
      • Baraniak J.
      • Frey P.A.
      Effect of ion pairing on bond order and charge localization in alkyl phosphorothioates.
      ). The diffusion coefficient and behavior on filtration confirm the claim that ss PS ASOs behave like flexible rods (
      • Plumridge A.
      • Meisburger S.P.
      • Andresen K.
      • Pollack L.
      The impact of base stacking on the conformations and electrostatics of single-stranded DNA.
      ). Molecular modeling demonstrates several interesting characteristics of these molecules, including substantial flexibility and partial helical structures. However, no modeling studies have been reported for PS containing ASOs or the chimeric PS ASOs that are broadly used today, but it is highly likely that PS ASOs behave similarly to ss oligonucleotides in solution. ASOs have a hydrophilic surface, the PS or PO linkages and a hydrophobic surface, the nucleobases, and thus are relatively amphipathic, an important property that affects protein binding and consequently distribution and cell uptake (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ). The nucleobases in an ss ASO are free to engage in Watson–Crick hybridization, hydrophobic, and base stacking interactions with nucleic acids and amino acids.
      A ds ASO (siRNA) is, of course, at least double the molecular weight of a comparable ss ASO. In an siRNA, the sense strand meets the formal definition of a drug delivery device; it is noncovalently bound, enhances the stability of the antisense strand, and must be removed by the AGO2 loading complex before the pharmacophore, the antisense strand, is active (
      • Setten R.L.
      • Rossi J.J.
      • Han S.P.
      The current state and future directions of RNAi-based therapeutics.
      ,
      • Chernikov I.V.
      • Vlassov V.V.
      • Chernolovskaya E.L.
      Current development of siRNA bioconjugates: From research to the clinic.
      ,
      • Manoharan M.
      • Rajeev K.G.
      Utilizing chemistry to harness RNA interference pathways for therapeutics: Chemically modified siRNAs and antagomirs.
      ,
      • Fougerolles A.R.D.
      • Maraganore J.M.
      Discovery and development of RNAi therapeutics.
      ). To date, very little information is available on waters of hydration, counterions, or diffusion coefficients for chemically modified ds ASOs, though a 2ʹ F ds RNA was shown to have limited hydration (
      • Manoharan M.
      • Akinc A.
      • Pandey R.K.
      • Qin J.
      • Hadwiger P.
      • John M.
      • Mills K.
      • Charisse K.
      • Maier M.A.
      • Nechev L.
      • Greene E.M.
      • Pallan P.S.
      • Rozners E.
      • Rajeev K.G.
      • Egli M.
      Unique gene-silencing and structural properties of 2'-fluoro- modified siRNAs.
      ), but it is reasonable to conclude that these agents are also rod-like, but with wider transverse dimensions and less flexibility than an ss ASO. More importantly, ds ASOs present two hydrophilic faces and the nucleobases are not free to engage in the stacking, hydrogen bonding, or hydrophobic interactions that are available to ss ASOs. These fundamental physicochemical properties are reflected in quite significant differences in the pharmacokinetic behavior of ss PS ASOs compared with ds PS containing siRNAs, which will be discussed in the pharmacokinetics section.

      Oligonucleotide medicinal chemistry

      As summarized above, the evolution of the medicinal chemistry of oligonucleotides has been critical to the steadily improving performance of ASOs in the clinic. More recently, based on understanding the importance of protein binding to the in vivo distribution, cell uptake, and subcellular distribution, focus has shifted to targeted delivery to specific cell types and organ systems and point modifications that alter interactions with specific cellular proteins (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ). Two phosphate modifications have proven to be most valuable, PMO (phosphorodiamidate morpholino oligomer) and PS. The PMO modification is neutral and is used in ASOs that have been shown to be very modestly active at high doses only in the treatment of patients with Duchenne’s muscular dystrophy (DMD), a disease of skeletal muscles (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ,
      • Iversen P.L.
      Morpholinos.
      ,
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ). The PS modification has proven far more important and is broadly used in all major classes of ASOs and all chemically modified siRNAs (Fig. 2A).
      Figure thumbnail gr2
      Figure 2Oligonucleotide chemical modifications. A, commonly used backbone modifications. MOP, methoxypropyl phosphonate; PMO, phosphorodiamidate morpholino oligomer; PO, phosphodiester; PS, phosphorothioate. B, commonly used 2’ modifications. cEt, constrained ethyl; F, fluoro; LNA, locked nucleic acid; Me, 2’-O-methyl; MOE, methoxyethyl. C, ASO designs. Fully modified ASOs that do not activate RNase H1 are commonly used to modulate splicing and translation. Gapmer ASOs that contain DNA gap and activate RNase H1 are normally modified at both ends of ASOs with different 2’ modifications. To further improve ASO performance, 2’Me or MOP (methoxypropyl phosphonate) modification is introduced into certain positions of the gap region.
      The replacement of one nonbridging oxygen with a sulfur alters the physicochemical characteristics of the phosphate in important ways. This modification results in the creation of a chiral center at each internucleotide link, so not surprisingly, the effects of the chiral centers have been extensively studied, as have the effects of pure R or S isomers (
      • Wan W.B.
      • Migawa M.T.
      • Vasquez G.
      • Murray H.M.
      • Nichols J.G.
      • Gaus H.
      • Berdeja A.
      • Lee S.
      • Hart C.E.
      • Lima W.F.
      • Swayze E.E.
      • Seth P.P.
      Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages.
      ,
      • Ostergaard M.E.
      • De Hoyos C.L.
      • Wan W.B.
      • Shen W.
      • Low A.
      • Berdeja A.
      • Vasquez G.
      • Murray S.
      • Migawa M.T.
      • Liang X.H.
      • Swayze E.E.
      • Crooke S.T.
      • Seth P.P.
      Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides.
      ,
      • Koziolkiewicz M.
      • Krakowiak A.
      • Kwinkowski M.
      • Boczkowska M.
      • Stec W.J.
      Stereodifferentiation--the effect of P chirality of oligo(nucleoside phosphorothioates) on the activity of bacterial RNase H.
      ,
      • Koziolkiewicz M.
      • Wojcik M.
      • Kobylanska A.
      • Karwowski B.
      • Rebowska B.
      • Guga P.
      • Stec W.J.
      Stability of stereoregular oligo(nucleoside phosphorothioate)s in human plasma: Diastereoselectivity of plasma 3'-exonuclease.
      ,
      • Jahns H.
      • Roos M.
      • Imig J.
      • Baumann F.
      • Wang Y.
      • Gilmour R.
      • Hall J.
      Stereochemical bias introduced during RNA synthesis modulates the activity of phosphorothioate siRNAs.
      ). Suffice it to say that no reproducible systemic advantages for the use of chirally pure PS ASOs have been identified. The issue of chirality will be further developed as the results of recent clinical studies with chirally pure PS ASOs are discussed a bit later in this review. Because the sulfur atom is twice as large as the oxygen atom, the charge distribution, bond angles, and stretching of PS links differ substantially from PO linkages (
      • Baraniak J.
      • Frey P.A.
      Effect of ion pairing on bond order and charge localization in alkyl phosphorothioates.
      ,
      • Steinke C.A.
      • Reeves K.K.
      • Powell J.W.
      • Lee S.A.
      • Chen Y.Z.
      • Wyrzykiewicz T.
      • Griffey R.H.
      • Mohan V.
      Vibrational analysis of phosphorothioate DNA: II. The POS group in the model compound dimethyl phosphorothioate [(CH3O)2(POS)].
      ). Put simply, the sulfur substitution spreads the charge and makes the phosphate more “lipophilic,” thereby facilitating binding to proteins. As a general rule, for proteins that require PS moieties to bind ASOs, the minimum number of this modification needed to support meaningful protein interactions is 10. In ASOs that contain both PS and PO moieties, the placement of PS units plays an important role, but, to date, no general rules have emerged, although PS placed at 5ʹ end of ASOs tends to bind more proteins and have higher affinities for key proteins (
      • Liang X.H.
      • Sun H.
      • Shen W.
      • Crooke S.T.
      Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages.
      ,
      • Liang X.H.
      • Shen W.
      • Sun H.
      • Kinberger G.A.
      • Prakash T.P.
      • Nichols J.G.
      • Crooke S.T.
      Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.
      ). The enhanced protein binding enabled by PS substitutions is critical as protein binding of ss PS ASOs plays crucial roles in the absorption, distribution, cellular uptake, intracellular distribution, activity, and toxicity of PS ASOs. This topic will be discussed in detail in a later section.
      Appropriate modifications of the 2ʹ-position in the ribose result in “pre-organization” of the RNA-PS ASO heteroduplex and thus enhance the affinity for cognate sequences in target RNAs. Commonly used 2ʹ modifications are listed in Figure 2B. This in turn increases the potency of PS ASOs, reducing the doses required to produce pharmacological effects (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ). Some 2ʹ modifications, e.g., 2ʹ-MOE, also substantially reduce proinflammatory effects (
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ,
      • Henry S.P.
      • Kim T.-W.
      • Karmer-Stickland K.
      • Zanardi T.A.
      • Fey R.A.
      • Levin A.A.
      Toxicologic properties of 2'-O-methoxyethyl chimeric antisense inhibitors in animals and man.
      ).
      More recently, focus has shifted to target delivery to specific tissues and cells. To date, the major success is the conjugation of GalNAc moieties to PS ASOs and siRNAs to enhance productive delivery to hepatocytes (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Setten R.L.
      • Rossi J.J.
      • Han S.P.
      The current state and future directions of RNAi-based therapeutics.
      ,
      • Tang X.
      • Zhang S.
      • Fu R.
      • Zhang L.
      • Huang K.
      • Peng H.
      • Dai L.
      • Chen Q.
      Therapeutic prospects of mRNA-based gene therapy for glioblastoma.
      ). On average, this modification increases the potency of PS ASO with 2ʹ-MOE or cEt modifications 15- to 30-fold in humans (
      • Crooke S.T.
      • Baker B.F.
      • Pham N.C.
      • Hughes S.G.
      • Kwoh T.J.
      • Cai D.
      • Tsimikas S.
      • Geary R.S.
      • Bhanot S.
      The effects of 2'-O-methoxyethyl oligonucleotides on renal function in humans.
      ). More recently, encouraging data have been published showing that a GLP-1 peptide conjugated to PS ASOs effectively delivered PS ASOs for the first time to beta cells of the pancreas (
      • Ammala C.
      • Drury 3rd, W.J.
      • Knerr L.
      • Ahlstedt I.
      • Stillemark-Billton P.
      • Wennberg- Huldt C.
      • Andersson E.M.
      • Valeur E.
      • Jansson-Lofmark R.
      • Janzen D.
      • Sundstrom L.
      • Meuller J.
      • Claesson J.
      • Andersson P.
      • Johansson C.
      • et al.
      Targeted delivery of antisense oligonucleotides to pancreatic beta-cells.
      ). Work continues to evaluate other ligands to enhance productive delivery of PS ASOs and siRNAs to other tissues and cells (
      • Wang S.
      • Allen N.
      • Prakash T.P.
      • Liang X.H.
      • Crooke S.T.
      Lipid conjugates enhance endosomal release of antisense oligonucleotides into cells.
      ,
      • Ostergaard M.E.
      • Jackson M.
      • Low A.
      • Chappell A.E.
      • Lee R.G.
      • Peralta R.Q.
      • Yu J.
      • Kinberger G.A.
      • Dan A.
      • Carty R.
      • Tanowitz M.
      • Anderson P.
      • Kim T.W.
      • Fradkin L.
      • Mullick A.E.
      • et al.
      Conjugation of hydrophobic moieties enhances potency of antisense oligonucleotides in the muscle of rodents and non-human primates.
      ,
      • Prakash T.P.
      • Mullick A.E.
      • Lee R.G.
      • Yu J.
      • Yeh S.T.
      • Low A.
      • Chappell A.E.
      • Ostergaard M.E.
      • Murray S.
      • Gaus H.J.
      • Swayze E.E.
      • Seth P.P.
      Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle.
      ).

      Architectural features of ASOs

      ASOs are oligomeric and comprised of nucleotide analogs. Because ASOs can be designed to work through a variety of post-RNA-binding mechanisms, numerous designs have been evaluated. As new molecular mechanisms of action are identified and new insights into the molecular mechanisms of distribution, cellular uptake and subcellular distributions, and various toxicities are reported, the designs are becoming progressively more complex. For example, ASOs of various motifs and compositions including various 2ʹ modifications, pendant groups, 5ʹ and 3ʹ termini and ever, more frequently, select modifications of specific nucleotides are being incorporated (
      • Shen W.
      • De Hoyos C.L.
      • Migawa M.T.
      • Vickers T.A.
      • Sun H.
      • Low A.
      • Bell 3rd, T.A.
      • Rahdar M.
      • Mukhopadhyay S.
      • Hart C.E.
      • Bell M.
      • Riney S.
      • Murray S.F.
      • Greenlee S.
      • Crooke R.M.
      • et al.
      Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.
      ,
      • Migawa M.T.
      • Shen W.
      • Wan W.B.
      • Vasquez G.
      • Oestergaard M.E.
      • Low A.
      • De Hoyos C.L.
      • Gupta R.
      • Murray S.
      • Tanowitz M.
      • Bell M.
      • Nichols J.G.
      • Gaus H.
      • Liang X.H.
      • Swayze E.E.
      • et al.
      Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins.
      ).

      Length

      A question that was answered early in the development of antisense technology was: what is the optimal length of an ASO? Theoretical calculations suggested that approximately 16 nucleotides should result in sufficient affinity to bind a cognate sequence and sufficient specificity for an ASO to affect a single transcript (
      • Crooke S.T.
      Antisense drug technology: Principles, strategies, and applications.
      ). The theoretical calculations were confirmed experimentally (
      • Monia B.P.
      • Johnston J.F.
      • Geiger T.
      • Muller M.
      • Fabbro D.
      Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-Raf kinase.
      ). Affinity and specificity vary, as a function of not only the length of the ASO but also the affinity per nucleotide for the cognate sequence. Thus, as new higher-affinity modifications were introduced, the length required to achieve binding to a target RNA and induce pharmacological effects was reduced. In fact, a 2ʹ-MOE chimeric PS ASO of 12 nucleotides was shown to effectively reduce a target expressed in renal proximal convolutes tubules, sodium-glucose cotransmitter 2 (SGLT2), in multiple species and to induce changes in glomerular filtration rate (GFR) in man (
      • Zanardi T.A.
      • Han S.C.
      • Jeong E.J.
      • Rime S.
      • Yu R.Z.
      • Chakravarty K.
      • Henry S.P.
      Pharmacodynamics and subchronic toxicity in mice and monkeys of ISIS 388626, a second-generation antisense oligonucleotide that targets human sodium glucose cotransporter 2.
      ,
      • van Meer L.
      • Moerland M.
      • van Dongen M.
      • Goulouze B.
      • de Kam M.
      • Klaassen E.
      • Cohen A.
      • Burggraaf J.
      Renal effects of antisense-mediated inhibition of SGLT2.
      ). However, the specificity for the cognate sequence versus all other RNAs varies inversely with length to at least 18 to 20 nucleotides. Theoretical analyses and experimental results have shown that higher-affinity PS ASOs have optimal affinity and specificity between 16 and 18 nucleotides (
      • Freier S.M.
      • Watt A.T.
      Basic principles of antisense drug discovery.
      ). The propensity of PS ASOs to form self-structures that inhibit binding to cognate sequences and other effects increases when ASO length exceeds 20 to 22 nucleotides, resulting in current approaches being generally limited to 16 to 20 nucleotides (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      Antisense drug technology: Principles, strategies, and applications.
      ).

      Designs of ASOs

      PS ASOs designed to bind to sites involved in the processing, translation, or degradation of target RNAs are typically fully 2ʹ modified (Fig. 2C). The 2ʹ modifications are used to enhance affinity for the target RNA and increase stability against nucleases, the enzymes that degrade PS ASOs (PMOs are used to alter splicing as well), and to avoid creating a heteroduplex that is a substrate for enzymes that degrade the RNA in a DNA/RNA-like duplex (RNase H1) or enzymes that degrade the RNA in an RNA/RNA-like duplex (AGO2) (
      • Crooke S.T.
      • Vickers T.A.
      • Lima W.F.
      • Wu H.-J.
      Mechanisms of antisense drug action, an introduction.
      ,
      • Sazani P.
      • Graziewicz M.A.
      • Kole R.
      Splice switching oligonucleotides as potential therapeutics.
      ,
      • Hodges D.
      • Crooke S.T.
      Inhibition of splicing of wild-type and mutated luciferase-adenovirus pre-mRNAs by antisense oligonucleotides.
      ). Any 2ʹ modification or neutral backbone at the nucleotides involved the enzymatic mechanism for either RNase H1 or AGO2 inhibits those enzymes (
      • Sigova A.
      • Zamore O.D.
      Small RNA silencing pathways.
      ,
      • Lima W.
      • Wu H.
      • Crooke S.T.
      The RNase H mechanism.
      ). Though, at first blush, designing ASOs for occupancy-only mediated mechanisms of action is straightforward, as new occupancy-only mechanisms are discovered, PS ASO designs have become more complex. For example, the affinity of an ASO designed to enhance the translation of a protein by binding to an upstream reading frame (uORF) translation initiation codon or a translation inhibitory element (TIE) must be “fine-tuned” so that it prevents the translation initiation complex from binding effectively to the upstream translation initiation site or to bind to and disrupt the structure of a TIE, yet be removed by helicases such that translation read through can occur (
      • Liang X.H.
      • Sun H.
      • Shen W.
      • Wang S.
      • Yao J.
      • Migawa M.T.
      • Bui H.H.
      • Damle S.S.
      • Riney S.
      • Graham M.J.
      • Crooke R.M.
      • Crooke S.T.
      Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.
      ,
      • Liang X.-h.
      • Shen W.
      • Sun H.
      • Migawa M.T.
      • Vickers T.A.
      • Crooke S.T.
      Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames.
      ). Therefore, these ASOs may include fewer PS moieties, lower-affinity 2ʹ modifications such as 2ʹ methoxy and at the 3ʹ terminus, higher-affinity nucleotides such as cEts. In contrast to PS ASOs that are designed to bind to uORFs and TIEs, which are located in the 5ʹ UTR of mRNAs, to exploit the No-Go Decay pathway, PS ASOs must be fully 2ʹ modified and of high affinity, but must bind in the coding region of the RNA (
      • Liang X.H.
      • Nichols J.G.
      • Hsu C.W.
      • Vickers T.A.
      • Crooke S.T.
      mRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway.
      ).
      In contrast, PS ASOs designed to cause degradation of target RNAs by creating an ASO-RNA duplex that is a substrate for a nuclease (as opposed to a complex pathway such as the No-Go Decay pathway) must meet the specific requirements imposed by the enzyme. RNase H1 requires a minimum of deoxynucleotides and charged phosphate analogs in the region of the ASO that participates in the enzymatic mechanism. Since the enzyme behaves essentially as a pair of calipers, the entire enzyme that measures the number of nucleotides per helical turn (this is defined by whether the duplex is DNA/DNA, RNA/RNA, or DNA/RNA-like) and the catalytic domain measures the width of the minor groove and senses whether the phosphate and 2ʹ OH contributed by the ASO are present (
      • Lima W.
      • Wu H.
      • Crooke S.T.
      The RNase H mechanism.
      ). PS ASO must be designed to meet the enzymes requirements, and this has led to the “gapmer” design in which various 2ʹ and phosphate modifications can be used in the “wings,” but only 2ʹ OH and PS or PO moieties are tolerated in the portion of the ASO (the central gap) that participates in the catalytic mechanism of RNase H1. Thus “gapmers” can tolerate a wide range of modifications in the wing and modifications at specific nucleotides in the gap can be employed to make the cleavage selective for an even more specific site or mutation and to alter interactions with proteins to enhance the therapeutic index of PS ASOs (
      • Shen W.
      • De Hoyos C.L.
      • Migawa M.T.
      • Vickers T.A.
      • Sun H.
      • Low A.
      • Bell 3rd, T.A.
      • Rahdar M.
      • Mukhopadhyay S.
      • Hart C.E.
      • Bell M.
      • Riney S.
      • Murray S.F.
      • Greenlee S.
      • Crooke R.M.
      • et al.
      Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.
      ,
      • Migawa M.T.
      • Shen W.
      • Wan W.B.
      • Vasquez G.
      • Oestergaard M.E.
      • Low A.
      • De Hoyos C.L.
      • Gupta R.
      • Murray S.
      • Tanowitz M.
      • Bell M.
      • Nichols J.G.
      • Gaus H.
      • Liang X.H.
      • Swayze E.E.
      • et al.
      Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins.
      ,
      • Ostergaard M.E.
      • Southwell A.L.
      • Kordasiewicz H.
      • Watt A.T.
      • Skotte N.H.
      • Doty C.N.
      • Vaid K.
      • Villanueva E.B.
      • Swayze E.E.
      • Bennett C.F.
      • Hayden M.R.
      • Seth P.P.
      Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS.
      ). Designing ss PS ASOs to create heteroduplexes that are substrates for AGO2 is much more complex because of the structural requirements of AGO2 and components of the loading complex, specifically Dicer. In the end, by alternating 2ʹF and 2ʹOMe nucleosides, placing PS modifications only at sites tolerated by AGO2 and employing a 3ʹ adenosine dinucleotide to optimize interactions with Dicer and a stable phosphate analog, 5ʹ vinyl (E) phosphonamidite to bind to the phosphate binding pocket of AGO2 were ss ASOs active in vivo (
      • Lima W.F.
      • Prakash T.P.
      • Murray H.M.
      • Kinberger G.A.
      • Li W.
      • Chappell A.E.
      • Li C.S.
      • Murray S.F.
      • Gaus H.
      • Seth P.P.
      • Swayze E.E.
      • Crooke S.T.
      Single-stranded siRNAs activate RNAi in animals.
      ). Once again, as new insights accumulate, the designs of ASOs to exploit various mechanisms of action become more complex and contain ever more site-specific modifications and cell and tissue targeting ligands (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ).

      Properties of ASOs currently being evaluated

      PMO ASOs

      Pharmacokinetics

      PMOs are readily absorbed after subcutaneous (SQ) administration, but to demonstrate even a modest pharmacological effect in the clinic, these two agents are dosed weekly at 30 to 80 mg/kg, making SQ dosing unattractive. All three approved drugs are administered as IV infusions. PMOs are readily absorbed after SQ dosing (t1/2 absorption: 30–120 min). Though they do not bind plasma proteins (or other proteins), they have a plasma elimination half-life of approximately 2 to 15 h (
      • Frank D.E.
      • Schnell F.J.
      • Akana C.
      • El-Husayni S.H.
      • Desjardins C.A.
      • Morgan J.
      • Charleston J.S.
      • Sardone V.
      • Domingos J.
      • Dickson G.
      • Straub V.
      • Guglieri M.
      • Mercuri E.
      • Servais L.
      • Muntoni F.
      • et al.
      Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy.
      ,
      • Cirak S.
      • Arechavala-Gomeza V.
      • Guglieri M.
      • Feng L.
      • Torelli S.
      • Anthony K.
      • Abbs S.
      • Garralda M.E.
      • Bourke J.
      • Wells D.J.
      • Dickson G.
      • Wood M.J.
      • Wilton S.D.
      • Straub V.
      • Kole R.
      • et al.
      Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: An open-label, phase 2, dose-escalation study.
      ). PMOs distribute to peripheral tissues, notably skeletal muscle, but detailed analyses of tissue concentrations have not been published. They have been reported to have tissue elimination half-lives of 7 to 14 days, and the main route of elimination is as intact molecules in urine (
      • Iversen P.L.
      Morpholinos.
      ,
      • Cirak S.
      • Arechavala-Gomeza V.
      • Guglieri M.
      • Feng L.
      • Torelli S.
      • Anthony K.
      • Abbs S.
      • Garralda M.E.
      • Bourke J.
      • Wells D.J.
      • Dickson G.
      • Wood M.J.
      • Wilton S.D.
      • Straub V.
      • Kole R.
      • et al.
      Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: An open-label, phase 2, dose-escalation study.
      ).

      Pharmacodynamics

      Because the internucleotide linkage in PMO ASOs is neutral and RNase H1 requires a phosphate or charged phosphate analog at internucleotide linkages near the site of cleavage of RNA and because RNase H1 requires an RNA/DNA-like helical conformation and minor groove dimensions, PMOs do not support RNase H1 cleavage (
      • Iversen P.L.
      Morpholinos.
      ). Though to date no studies of PMO ASOs or PMO-containing siRNAs have been published, given the structural requirements to support AGO2 cleavage, it is highly unlikely that PMOs would support AGO2 cleavage (
      • Sigova A.
      • Zamore O.D.
      Small RNA silencing pathways.
      ). Thus, in principle, PMO ASOs can induce pharmacological effects only via occupancy-only mechanisms such as a steric block of translation or alterations in splicing (
      • Iversen P.L.
      Morpholinos.
      ). To date, the only mechanism to display consistent activity in mammalian cells has been the alteration of splicing (
      • Iversen P.L.
      Morpholinos.
      ). In fact, the only transcript to be shown to be amenable to shifts in splicing due to PMOs is dystrophin. The sole tissue in which consistent pharmacological effects have been demonstrated is skeletal muscle, and the only disease to show even modest consistent benefit is DMD. Thus, in mammalian systems, PMO ASOs have demonstrated very limited applicability.

      Toxicology

      In both animals and humans, PMO ASOs have been shown to have excellent safety profiles.

      Eteplirsen

      To date, three PMO ASOs have been approved for commercial use in a major market (Table 1). All three of these PMO ASOs target the dystrophin transcript, work via the same mechanism, have similar pharmacokinetics and potencies, and are used to treat the same disease, DMD (Table 1). Consequently, we will focus on eteplirsen, perhaps the best characterized of the three provides a solid perspective on the performance of PMOs in the clinic.
      DMD is an X-linked genetic disease caused by mutations in the dystrophin gene, a large gene with multiple introns and in which a wide range of mutations have been observed. Dystrophin is an essential component of the sarcolemma. Thus, all skeletal muscles are adversely affected by loss-of-function mutations in dystrophin. The disease typically is manifested around ages 3 to 5 and progresses where patients are confined to wheelchairs as teenagers often leading to death in the 20s. As all skeletal muscles are affected, pulmonary function also declines with age.
      Given the observations that some truncation mutations in the dystrophin gene result in partially functional truncated protein, the PMO strategy is to cause skipping of an exon that results in a coding sequence that can be translated into a partially functional partial protein. About 14% of DMD patients have mutations that are amenable to exon 51 skipping resulting in a functional partial coding sequence and partial protein. Eteplirsen is designed to cause exon 51 skipping. It has been evaluated in several clinical trials in ambulatory DMD patients typically at 30 to 50 mg/kg weekly as an IV infusion. In shorter-term trials, evidence of increased dystrophin levels was achieved, but little evidence of clinical benefit was observed. However, longer-term studies have confirmed the increase in dystrophin protein and modest clinical benefit. In ambulatory patients, a 6-min walk test is usually performed. Upper limb strength is also evaluated and is the main efficacy measure in nonambulatory patients. Though eteplirsen resulted in a modest reduction in the rate of loss in skeletal muscle performance in a 6-min walk test, the differences were statistically significant and even in patients who became nonambulatory, upper limb strength and cardiac performance were stabilized (
      • Alfano L.N.
      • Charleston J.S.
      • Connolly A.M.
      • Cripe L.
      • Donoghue C.
      • Dracker R.
      • Dworzak J.
      • Eliopoulos H.
      • Frank D.E.
      • Lewis S.
      • Lucas K.
      • Lynch J.
      • Milici A.J.
      • Flynt A.
      • Naughton E.
      • et al.
      Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.
      ). In other studies, eteplirsen also reduced the progression in loss of lung function (
      • Khan N.
      • Eliopoulos H.
      • Han L.
      • Kinane T.B.
      • Lowes L.P.
      • Mendell J.R.
      • Gordish- Dressman H.
      • Henricson E.K.
      • McDonald C.M.
      • Eteplirsen I.
      • the C.D.I.
      Eteplirsen treatment attenuates respiratory decline in ambulatory and non- ambulatory patients with Duchenne muscular dystrophy.
      ). Thus, for a select group of DMD patients, this drug appears to be safe and effective therapy. The approval of eteplirsen was controversial because the phase 3 trial failed to provide statistically compelling evidence of efficacy. Furthermore, the FDA required that the manufacturer note that production of functional protein has not been proven in the label. Only a small fraction of DMD patients have this specific mutation.

      Impact of the PS modification on antisense technology

      With the single exception of PMO drugs, all currently employed ASOs and all chemically modified siRNAs contain at least some PS linkages and, in many cases, PS moieties at all internucleotide links (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ). The importance of PS modifications to RTDs cannot be overstated for without this modification, it is unlikely that RTD technology would exist today. Fortunately, as a result of progress reported in the past few years, we now have detailed molecular mechanisms to explain the effects of PS modified RTDs on their interactions in biological systems (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ). These will be discussed in subsequent sections of this review. In this section, we will describe the properties of the various chemical classes of PS ASOs.

      Chiral PS ASOs

      The substitution of sulfur for a nonbridging oxygen in a phosphate group creates a chiral center at every PS linkage (referred to as Sp and Rp), so in a fully phosphorylated 20 mer compound, there are 219 Rp or Sp stereoisomers. Quite naturally, significant efforts have been invested in developing stereochemical synthetic schemes and in the evaluation of the effect of chirality on the behavior of PS ASOs in biological systems (
      • Levin A.A.
      • Yu R.Z.
      • Geary R.S.
      Basic principles of the pharmaseokinetics of antisense oligonucleotide drugs.
      ,
      • Eckstein F.
      Phosphorothioate oligodeoxynucleotides: What is their origin and what is unique about them?.
      ,
      • Oka N.
      • Wada T.
      • Saigo K.
      An oxazaphospholidine approach for the stereocontrolled synthesis of oligonucleoside phosphorothioates.
      ). It is thought that the increased nuclease resistance of chirally impure PS ASOs is due to the sulfur displacing the divalent cation that most nucleases use as a part of the enzymatic mechanism from the catalytic center of the enzymes. Alternatively, the sulfur may be less able to take on a negative charge during the reaction (
      • Levin A.A.
      • Yu R.Z.
      • Geary R.S.
      Basic principles of the pharmaseokinetics of antisense oligonucleotide drugs.
      ). In either case, it is sensible to assume that the Rp and Sp stereoisomers might differ in their sensitivity to nucleases, and indeed, that is the case. The Sp isomer is more nuclease-resistant than the Rp isomer (
      • Wan W.B.
      • Migawa M.T.
      • Vasquez G.
      • Murray H.M.
      • Nichols J.G.
      • Gaus H.
      • Berdeja A.
      • Lee S.
      • Hart C.E.
      • Lima W.F.
      • Swayze E.E.
      • Seth P.P.
      Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages.
      ). So, an Sp chirally pure PS ASO is more stable to nucleases than the Rp chirally pure ASO. Similarly, it is logical to assume that the stereoisomers might differ in their effects on hybridization of the PS ASO and the cognate RNA sequence, and once again the assumption is correct. Unfortunately, and not surprisingly, the Rp stereoisomer results in improved affinity per Rp linkage compared with the Sp or a diastereomeric chiral mixture. Thus, what is “seen” as the more “natural” nucleic acid hybridizes to the cognate sequence with higher affinity and is readily degraded by nucleases. This means that a chirally impure diastereomeric PS ASO might bind to the target RNA with somewhat lower affinity than natural DNA and be more nuclease-resistant, and that is what is observed.
      Many PS ASOs are designed to activate RNase H1 and therefore are chimeric with wings comprised of 2ʹ modified sugars and a PS oligodeoxynucleotide gap (
      • Crooke S.T.
      • Vickers T.A.
      • Lima W.F.
      • Wu H.-J.
      Mechanisms of antisense drug action, an introduction.
      ). Since PS ASOs interact with RNase H1 at both the hybrid binding domain and in the catalytic center and the enzyme displays sequence preferences for cleavage, one would expect that the overall effects of chiral purity on activity and toxicity might be complex, and indeed they are. But the bottom line is this: thorough studies to evaluate the overall impact of chiral purity on activity and toxicity have shown that chiral purity or chiral purity at individual PS units provides no meaningful improvement in therapeutic index and overall performance of PS ASOs (
      • Ostergaard M.E.
      • De Hoyos C.L.
      • Wan W.B.
      • Shen W.
      • Low A.
      • Berdeja A.
      • Vasquez G.
      • Murray S.
      • Migawa M.T.
      • Liang X.H.
      • Swayze E.E.
      • Crooke S.T.
      • Seth P.P.
      Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides.
      ). Clinical results to date appear to reflect that theoretical analysis.

      Huntington’s disease (HD)

      WVE-120101 and WVE-120102 are chirally pure PS 2ʹ methoxy gapmer ASOs that target the mutant forms of Huntingtin (HTT), rs362307 and rs362331, respectively, which are currently under evaluation in two separate phase 1 b/2a studies (
      • Panzara M.
      Selective Targeting of Mutant Huntingtin Using Stereopure Oligonucleotides as a Potential Therapeutic Approach for Huntington’s Disease.
      ).
      WVE-120102 was administered intrathecally at doses of 2 to 16 mg every 2 months to patients with HD. An interim analysis was presented. No safety or tolerability data were shown, but the drug was reported to be safe and well tolerated. A comparison of all patients treated at any dose level showed a 12.4% reduction in mutant HTT protein in the CSF, achieving a p-value <0.05, and an analysis of the effects on HTT protein at each dose level suggested a dose–response relationship. The study is now enrolling HD patients in the 32 mg every 2-week cohort (
      Wave Life Sciences
      Wave Life Sciences Announces Topline Data and Addition of Higher Dose Cohort in Ongoing Phase 1b/2a PRECISION-HD2 Trial in Huntington’s Disease.
      ).

      Duchenne’s muscular dystrophy

      Suvodirsen (WVE-210201), a fully 2ʹ methoxy, 2ʹ-F modified PS ASO with chirally pure PS moieties at selected sites designed to increase skipping of exon 51, was evaluated at doses of 0.5 to 10 mg/kg in a multidose 12-week study. While safe and well tolerated, the drug did not show increases in dystrophin protein in muscle in the phase 1 open-label extension with weekly IV doses of 3.5 or 5.0 mg/kg. Nor was there evidence of effects at any dose of any parameter tested (
      • Wagner K.
      • Phan H.
      • Servais L.
      • Gidaro T.
      • Cripe L.
      • Eagle M.
      • Muntoni F.
      • Straub V.
      • Lonkar P.
      • Schmitz S.
      • Lake S.
      • Hu X.
      • Panzara M.
      O.28Safety and tolerability of suvodirsen (WVE-210201) in patients with Duchenne muscular dystrophy: Results from a phase 1 clinical trial.
      ). Development of this PS ASO was subsequently terminated (
      Wave Life Sciences
      Wave Life Sciences Announces Discontinuation of Suvodirsen Development for Duchenne Muscular Dystrophy.
      ).

      2ʹ-O-methoxyethyl (2ʹ-MOE) PS ASOs

      2ʹ-MOE is one of several thousand 2ʹ modifications of the furanose evaluated (
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ). Obviously 2ʹ-MOE is substantially bulkier than hydrogen and is more hydrophilic and thus more hydrophilic. Because it fixes the furanose in a favorable conformation in effect “pre-organizing the duplex,” 2ʹ-MOE increases the Tm per nucleotide by more than 2 degrees. In a typical chimeric ASO designed to serve as a substrate for RNase H1, there is a central PS deoxynucleotide center of 8 to 10 nucleotides flanked by PS 2ʹ modified nucleotides. Thus, a typical “gapmer” ASO has 8 to 10 2ʹ-MOE nucleotides resulting an increase in affinity for cognate RNA sequences of several orders of magnitude (
      • Bennett C.F.
      Pharmacological properties of 2'-O-Methoxyethyl-Modified oligonucleotides.
      ). This, in turn translates into a 10- to 20-fold increase in potency compared with PS oligodeoxynucleotides in animal models. Because it is not clear that PS oligonucleotides administered systemically in humans resulted in antisense-mediated pharmacological effects, direct comparison with PS 2ʹ-MOE ASOs is difficult, but a 50- to 100-fold increase in potency in humans is a reasonable estimate. That the increase in affinity for the target RNA is not fully reflected in the increase in potency compared with PS oligodeoxynucleotides is explained by several factors. In a “gapmer” ASO, the potential substrate for RNase H1 is reduced to 8 to 10 nucleotides. Human RNase H1 requires at least four contiguous deoxynucleotides to cleave RNA in an ASO–RNA duplex, and enzyme activity is not optimal until about eight contiguous deoxynucleotides are present. Moreover, human RNase H1 (and all mammalian RNase H1 enzymes) displays sequence preferences. Therefore, the efficiency of the enzyme is substantially reduced (
      • Lima W.
      • Wu H.
      • Crooke S.T.
      The RNase H mechanism.
      ).
      The 2ʹ-MOE moiety provides additional significant advantages. It substantially increases resistance to nucleases, the enzymes that degrade PS ASOs. This is a result of the steric bulk of the substitution and its hydrophilic character (
      • Bennett C.F.
      Pharmacological properties of 2'-O-Methoxyethyl-Modified oligonucleotides.
      ). This translates into an increase in tissue elimination half-life from about 48 h to 2 to 4 weeks facilitating weekly to monthly systemic administration (
      • Bennett C.F.
      Pharmacological properties of 2'-O-Methoxyethyl-Modified oligonucleotides.
      ,
      • Geary R.S.
      • Yu R.Z.
      • Siwkoswki A.
      • Levin A.A.
      Pharmacokinetic/pharmacodynamic properties of phosphorothioate 2'-O-(2- methoxyethyl)-modified antisense oligonucleotides in animals and man.
      ). Additionally, 2ʹ-MOE substitution significantly reduces proinflammatory effects (
      • Henry S.P.
      • Kim T.-W.
      • Karmer-Stickland K.
      • Zanardi T.A.
      • Fey R.A.
      • Levin A.A.
      Toxicologic properties of 2'-O-methoxyethyl chimeric antisense inhibitors in animals and man.
      ,
      • Levin A.A.
      • Yu R.Z.
      • Geary R.S.
      Basic principles of the pharmaseokinetics of antisense oligonucleotide drugs.
      ). The molecular mechanisms responsible for the reduced inflammatory effects are not fully understood, but probably related to changes in interactions with toll-like receptors (TLRs), particularly TLR 9 (
      • Levin A.A.
      • Yu R.Z.
      • Geary R.S.
      Basic principles of the pharmaseokinetics of antisense oligonucleotide drugs.
      ,
      • Bennett C.F.
      Pharmacological properties of 2'-O-Methoxyethyl-Modified oligonucleotides.
      ).
      PS 2ʹ-MOE ASOs are, by far, the best characterized of any RTD chemical or structural class. More than 10,000 patients in clinical trials have been treated at doses that range from 50 mg to 1200 mg/weekly and treatment durations ranging from 6 weeks to >5 years. In the integrated safety database for 2ʹ-MOE ASOs, there are data from more than 8000 patients treated in randomized, placebo-controlled trials (
      • Crooke S.T.
      • Baker B.F.
      • Pham N.C.
      • Hughes S.G.
      • Kwoh T.J.
      • Cai D.
      • Tsimikas S.
      • Geary R.S.
      • Bhanot S.
      The effects of 2'-O-methoxyethyl oligonucleotides on renal function in humans.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Kwoh T.J.
      • Cheng W.
      • Schulz D.J.
      • Xia S.
      • Salgado N.
      • Bui H.H.
      • Hart C.E.
      • Burel S.A.
      • Younis H.S.
      • Geary R.S.
      • Henry S.P.
      • Bhanot S.
      Integrated safety assessment of 2'-O-methoxyethyl chimeric antisense oligonucleotides in NonHuman primates and healthy human volunteers.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Witztum J.L.
      • Kwoh T.J.
      • Pham N.C.
      • Salgado N.
      • McEvoy B.W.
      • Cheng W.
      • Hughes S.G.
      • Bhanot S.
      • Geary R.S.
      The effects of 2'-O-methoxyethyl containing antisense oligonucleotides on platelets in human clinical trials.
      ). Systemic delivery of PS 2ʹ-MOE ASOs is achieved after IV, SQ, and IM administration. Mipomersen, a PS 2ʹ-MOE gapmer ASO designed to reduce apoB-100, the key structural protein of LDL cholesterol, has also been administered orally to several species in enteric-coated (to prevent acid-dependent precipitation and depurination in the stomach) formulations containing decanoic acid (C10) as a penetration enhancer. In humans, approximately 6% oral bioavailability and statistically significant reductions of plasma apoB-100 and LDL cholesterol were observed in normal volunteers (
      • Tillman L.G.
      • Geary R.S.
      • Hardee G.E.
      Oral delivery of antisense oligonucleotides in man.
      ). The total dose and number of capsules required meant that this formulation was not appropriate for commercial use. However, the study provided important proof of principle and suggested that with more potent ASOs, commercially attractive oral dosing might be achievable. Administration for local therapy has been achieved after intravitreal (IVT), aerosol, intradermal, intrathecal (IT), and rectal administration (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ).
      The pharmacokinetics of PS MOE ASOs have been well studied in multiple species, including humans. After SQ or IM administration, these agents are rapidly and completely absorbed (t1/2 absorption approximately 60 min). They are protein bound in plasma (>90% at doses of 300 mg/week in man), and the main proteins in plasma to which they bind have been identified (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Gaus H.J.
      • Gupta R.
      • Chappell A.E.
      • Ostergaard M.E.
      • Swayze E.E.
      • Seth P.P.
      Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay.
      ). Albumin is the major protein that binds to the ASOs with a kd of approximately 13 μM, but they do not bind to the major drug-binding sites in that protein. They are then rapidly distributed to peripheral tissues with the liver, kidney, fat cells, and spleen accumulating the most drug at low doses. At higher doses, those tissues saturate and distribution to secondary tissues occurs. The suborgan distribution has also been characterized in the liver and kidney after systemic administration and the CNS and lung after local administration. They are cleared slowly from peripheral tissues with elimination half-lives of 2 to 4 weeks (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ,
      • Levin A.A.
      • Yu R.Z.
      • Geary R.S.
      Basic principles of the pharmaseokinetics of antisense oligonucleotide drugs.
      ,
      • Geary R.S.
      • Yu R.Z.
      • Siwkoswki A.
      • Levin A.A.
      Pharmacokinetic/pharmacodynamic properties of phosphorothioate 2'-O-(2- methoxyethyl)-modified antisense oligonucleotides in animals and man.
      ). Elimination after IT administration is limited by the flow of CSF and supports quarterly or less frequent dosing. Clearance from the lung after aerosol delivery is similar to other tissues after systemic administration and therefore, weekly aerosol dosing is typical. Systemic bioavailability after either IT or aerosol administration is limited and since the doses by either route are quite low, little systemic exposure results from ASOs delivered by either route. Elimination is due to endo- and exonuclease digestion with clearance of metabolites (oligonucleotide fragments) in urine (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ,
      • Levin A.A.
      • Yu R.Z.
      • Geary R.S.
      Basic principles of the pharmaseokinetics of antisense oligonucleotide drugs.
      ,
      • Geary R.S.
      • Yu R.Z.
      • Siwkoswki A.
      • Levin A.A.
      Pharmacokinetic/pharmacodynamic properties of phosphorothioate 2'-O-(2- methoxyethyl)-modified antisense oligonucleotides in animals and man.
      )
      After SQ administration, the ED50 for targets expressed in hepatocytes is about 150 mg/week in man. Somewhat higher doses are required for other tissues and doses correlate well with the distribution of PS 2ʹ-MOE ASOs. Dose–response curves are log-linear (
      • Bennett C.F.
      Pharmacological properties of 2'-O-Methoxyethyl-Modified oligonucleotides.
      ). Thus, these agents are straightforward to administer, and different PS ASOs of class behave quite similarly, which facilitates dose selection and development (
      • Bennett C.F.
      Pharmacological properties of 2'-O-Methoxyethyl-Modified oligonucleotides.
      ,
      • Geary R.S.
      • Yu R.Z.
      • Siwkoswki A.
      • Levin A.A.
      Pharmacokinetic/pharmacodynamic properties of phosphorothioate 2'-O-(2- methoxyethyl)-modified antisense oligonucleotides in animals and man.
      ). The safety profile is very well understood after administration by all routes of delivery (
      • Crooke S.T.
      Molecular mechanisms of antisense oligonucleotides.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Kwoh T.J.
      • Cheng W.
      • Schulz D.J.
      • Xia S.
      • Salgado N.
      • Bui H.H.
      • Hart C.E.
      • Burel S.A.
      • Younis H.S.
      • Geary R.S.
      • Henry S.P.
      • Bhanot S.
      Integrated safety assessment of 2'-O-methoxyethyl chimeric antisense oligonucleotides in NonHuman primates and healthy human volunteers.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Witztum J.L.
      • Kwoh T.J.
      • Pham N.C.
      • Salgado N.
      • McEvoy B.W.
      • Cheng W.
      • Hughes S.G.
      • Bhanot S.
      • Geary R.S.
      The effects of 2'-O-methoxyethyl containing antisense oligonucleotides on platelets in human clinical trials.
      ).
      All PS ASOs result in peak plasma-related transient inhibition of clotting, which has proven of no clinical significance. In nonhuman primates (NHP), they also result in peak plasma-related activation of the alternative complement cascade, but in humans this, once again, has not been an issue. All PS ASOs result in transient clinically insignificant reduction in platelet count of about 20 to 30%, which is also peak plasma concentration dependent. The most common adverse event in humans is injection site reactions (ISRs) after SQ administration that are dose-dependent and typically mild, but can be associated with discontinuation of treatment. At a dose of 300 mg/week, two PS 2ʹ-MOE ASOs, volanesorsen and inotersen, are associated with clinically significant thrombocytopenia in a few patients suffering from familial chylomicronemia (FCS) (
      • Witztum J.L.
      • Gaudet D.
      • Freedman S.D.
      • Alexander V.J.
      • Digenio A.
      • Williams K.R.
      • Yang Q.
      • Hughes S.G.
      • Geary R.S.
      • Arca M.
      • Stroes E.S.G.
      • Bergeron J.
      • Soran H.
      • Civeira F.
      • Hemphill L.
      • et al.
      Volanesorsen and triglyceride levels in familial chylomicronemia syndrome.
      ) and transthyretin amyloidosis (TTR amyloidosis). In TTR patients, the mechanism of thrombocytopenia appears to be related to ASO-dependent antiplatelet antibodies (
      • Narayanan P.
      • Curtis B.R.
      • Shen L.
      • Schneider E.
      • Tami J.A.
      • Paz S.
      • Burel S.A.
      • Tai L.J.
      • Machemer T.
      • Kwoh T.J.
      • Xia S.
      • Shattil S.J.
      • Witztum J.L.
      • Engelhardt J.A.
      • Henry S.P.
      • et al.
      Underlying immune disorder may predispose some transthyretin amyloidosis subjects to inotersen-mediated thrombocytopenia.
      ).

      PS 2ʹ S-constrained ethyl (cEt)

      PS 2ʹ S-constrained ethyl (cEts), also referred to as generation 2.5 PS ASOs, contain a bicyclic sugar that constrains the furanose surrogate to adopt a conformation favorable to hybridization to RNA. Therefore, the affinity per nucleotide for RNA is increased by approximately 2º in thermal melting experiments compared with PS 2ʹ-MOE ASOs. cEts are members of the bicyclic sugar modification class, several of which have been evaluated preclinically and in the clinic. Another 2ʹ bicyclic sugar, locked nucleic acid (LNA) results in even greater affinity per nucleotide than the cEt modification (
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ,
      • Koch T.
      • Orum H.
      Locked nucleic acid.
      ). The increase in affinity per nucleotide of the cEt modification leads to an approximately tenfold increase in potency. In addition to the obvious benefit of being able to employ lower doses in tissues that accumulate PS ASOs at low doses, in principle an increase in potency might enhance the pharmacological activity in tissues that accumulate less PS ASOs, such as various types of cancer or skeletal muscle. The likelihood that an increased activity in lower accumulating tissues is enhanced because the pharmacokinetic behaviors of 2ʹ PS MOE, PS 2ʹ-cEt, and PS 2ʹ LNA ASOs are quite similar. Indeed, this supposition has been proven to be true.
      Arguably, the best example of using an increase in potency to achieve better pharmacological activity in a tissue in which lower concentrations accumulate is various types of cancer. PS 2ʹ- MOE ASOs have been thoroughly evaluated in multiple cancer types against a variety of targets. At very high doses (>900 mg/week IV), limited pharmacological activity was observed is a few malignant cells, e.g., prostate or lymphoid tissues, but certainly insufficient to justify commercial approval (
      • Bennett C.F.
      Pharmacological properties of 2'-O-Methoxyethyl-Modified oligonucleotides.
      ). STAT3 2.5 LRx is a PS 2ʹ cEt targeting STAT3, a well-understood target necessary for survival of cancers. Broad phase 2 studies of this drug demonstrated meaningful antitumor activity in several cancers including head and neck (
      • MacLeod A.R.
      • Crooke S.T.
      RNA therapeutics in oncology: Advances, challenges, and future directions.
      ).
      However, the enhanced potency of PS cEt ASOs was associated with a significant increase in both the incidence and severity of cytotoxicity compared with PS 2ʹ-MOE ASOs in multiple cell lines in vitro and hepatotoxicity in mouse, rats, and NHP. PS LNA ASOs are another example of constrained carbohydrate containing ASOs. As mentioned previously, the LNA substitution results in approximately double the increase in Tm per nucleotide and thus was considered quite promising. Screening of PS LNA ASOs demonstrated an increase in the incidence and severity of cytotoxicity and hepatoxicity that was even greater than observed with PS cEt ASOs (
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ,
      • Koch T.
      • Orum H.
      Locked nucleic acid.
      ,
      • Burel S.A.
      • Hart C.E.
      • Cauntay P.
      • Hsiao J.
      • Machemer T.
      • Katz M.
      • Watt A.
      • Bui H.H.
      • Younis H.
      • Sabripour M.
      • Freier S.M.
      • Hung G.
      • Dan A.
      • Prakash T.P.
      • Seth P.P.
      • et al.
      Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.
      ,
      • Janssen H.L.
      • Reesink H.W.
      • Lawitz E.J.
      • Zeuzem S.
      • Rodriguez-Torres M.
      • Patel K.
      • van der Meer A.J.
      • Patick A.K.
      • Chen A.
      • Zhou Y.
      • Persson R.
      • King B.D.
      • Kauppinen S.
      • Levin A.A.
      • Hodges M.R.
      Treatment of HCV infection by targeting microRNA.
      ,
      • van der Ree M.H.
      • van der Meer A.J.
      • de Bruijne J.
      • Maan R.
      • van Vliet A.
      • Welzel T.M.
      • Zeuzem S.
      • Lawitz E.J.
      • Rodriguez-Torres M.
      • Kupcova V.
      • Wiercinska- Drapalo A.
      • Hodges M.R.
      • Janssen H.L.
      • Reesink H.W.
      Long-term safety and efficacy of microRNA-targeted therapy in chronic hepatitis C patients.
      ,
      • Swayze E.E.
      • Siwkowski A.M.
      • Wancewicz E.V.
      • Migawa M.T.
      • Wyrzykiewicz T.K.
      • Hung G.
      • Monia B.P.
      • Bennett C.F.
      Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.
      ). In humans, PS LNA ASOs caused severe hepatotoxicity, thrombocytopenia, and nephrotoxicity (
      • Janssen H.L.
      • Reesink H.W.
      • Lawitz E.J.
      • Zeuzem S.
      • Rodriguez-Torres M.
      • Patel K.
      • van der Meer A.J.
      • Patick A.K.
      • Chen A.
      • Zhou Y.
      • Persson R.
      • King B.D.
      • Kauppinen S.
      • Levin A.A.
      • Hodges M.R.
      Treatment of HCV infection by targeting microRNA.
      ,
      • Hagedorn P.H.
      • Persson R.
      • Funder E.D.
      • Albaek N.
      • Diemer S.L.
      • Hansen D.J.
      • Moller M.R.
      • Papargyri N.
      • Christiansen H.
      • Hansen B.R.
      • Hansen H.F.
      • Jensen M.A.
      • Koch T.
      Locked nucleic acid: Modality, diversity, and drug discovery.
      ). The apparent correlation between affinity increases beyond that provided by the 2ʹ-MOE substitution resulted in speculation that the toxicities of very high affinity PS ASOs is due to off-target hybridization and RNase H1 cleavage. Cleavage of very large transcripts was thought to be a principle driver of the toxicities because in very long transcripts, there is a higher probability of sequences that are near or perfect matches to the cognate sequence for which the ASO was designed (
      • Burel S.A.
      • Hart C.E.
      • Cauntay P.
      • Hsiao J.
      • Machemer T.
      • Katz M.
      • Watt A.
      • Bui H.H.
      • Younis H.
      • Sabripour M.
      • Freier S.M.
      • Hung G.
      • Dan A.
      • Prakash T.P.
      • Seth P.P.
      • et al.
      Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.
      ). When RNase H1 knockout mice were created and studied, the hypothesis was apparently confirmed because the hepatotoxicity of the very high affinity PS ASOs was substantially ameliorated (
      • Burel S.A.
      • Hart C.E.
      • Cauntay P.
      • Hsiao J.
      • Machemer T.
      • Katz M.
      • Watt A.
      • Bui H.H.
      • Younis H.
      • Sabripour M.
      • Freier S.M.
      • Hung G.
      • Dan A.
      • Prakash T.P.
      • Seth P.P.
      • et al.
      Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.
      ,
      • Lima W.F.
      • Murray H.M.
      • Damle S.S.
      • Hart C.E.
      • Hung G.
      • De Hoyos C.L.
      • Liang X.H.
      • Crooke S.T.
      Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function.
      ). However, additional work that will be discussed defined a step-by-step mechanism to explain the behavior of most toxic PS ASOs that is entirely unrelated to off-target hybridization (
      • Shen W.
      • De Hoyos C.L.
      • Migawa M.T.
      • Vickers T.A.
      • Sun H.
      • Low A.
      • Bell 3rd, T.A.
      • Rahdar M.
      • Mukhopadhyay S.
      • Hart C.E.
      • Bell M.
      • Riney S.
      • Murray S.F.
      • Greenlee S.
      • Crooke R.M.
      • et al.
      Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.
      ).

      PS -MOE GalNAc ASOs

      Targeted delivery to hepatocytes has had an important positive impact of PS ss ASOs and an even more profound influence on modified PS containing DS ASOs, siRNAs (
      • Crooke S.T.
      • Wang S.
      • Vickers T.A.
      • Shen W.
      • Liang X.H.
      Cellular uptake and trafficking of antisense oligonucleotides.
      ). The conjugation of a GalNAc to PS 2ʹ-MOE ASOs increases the potency of these agents in humans by 15- to 30-fold compared with the parent molecules by enhancing productive delivery of PS MOEASOs to hepatocytes without altering the overall distribution of these drugs to peripheral tissues (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Nair J.K.
      • Attarwala H.
      • Sehgal A.
      • Wang Q.
      • Aluri K.
      • Zhang X.
      • Gao M.
      • Liu J.
      • Indrakanti R.
      • Schofield S.
      • Kretschmer P.
      • Brown C.R.
      • Gupta S.
      • Willoughby J.L.S.
      • Boshar J.A.
      • et al.
      Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Pham N.C.
      • Hughes S.G.
      • Kwoh T.J.
      • Cai D.
      • Tsimikas S.
      • Geary R.S.
      • Bhanot S.
      The effects of 2'-O-methoxyethyl oligonucleotides on renal function in humans.
      ,
      • Prakash T.P.
      • Graham M.J.
      • Yu J.
      • Carty R.
      • Low A.
      • Chappell A.
      • Schmidt K.
      • Zhao C.
      • Aghajan M.
      • Murray H.F.
      • Riney S.
      • Booten S.L.
      • Murray S.F.
      • Gaus H.
      • Crosby J.
      • et al.
      Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N- acetyl galactosamine improves potency 10-fold in mice.
      ,
      • Viney N.J.
      • van Capelleveen J.C.
      • Geary R.S.
      • Xia S.
      • Tami J.A.
      • Yu R.Z.
      • Marcovina S.M.
      • Hughes S.G.
      • Graham M.J.
      • Crooke R.M.
      • Crooke S.T.
      • Witztum J.L.
      • Stroes E.S.
      • Tsimikas S.
      Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): Two randomised, double-blind, placebo-controlled, dose-ranging trials.
      ,
      • Graham M.J.
      • Lee R.G.
      • Brandt T.A.
      • Tai L.J.
      • Fu W.
      • Peralta R.
      • Yu R.
      • Hurh E.
      • Paz E.
      • McEvoy B.W.
      • Baker B.F.
      • Pham N.C.
      • Digenio A.
      • Hughes S.G.
      • Geary R.S.
      • et al.
      Cardiovascular and metabolic effects of ANGPTL3 antisense oligonucleotides.
      ,
      • Alexander V.J.
      • Xia S.
      • Hurh E.
      • Hughes S.G.
      • O'Dea L.
      • Geary R.S.
      • Witztum J.L.
      • Tsimikas S.
      N-acetyl galactosamine-conjugated antisense drug to APOC3 mRNA, triglycerides and atherogenic lipoprotein levels.
      ). Fortunately, it has been possible to compare the behavior of GalNAc conjugated PS 2ʹ-MOE ASOs to the behavior of the unconjugated ASO in several clinical trials, including large phase 2 studies (
      • Crooke S.T.
      • Baker B.F.
      • Pham N.C.
      • Hughes S.G.
      • Kwoh T.J.
      • Cai D.
      • Tsimikas S.
      • Geary R.S.
      • Bhanot S.
      The effects of 2'-O-methoxyethyl oligonucleotides on renal function in humans.
      ).
      To highlight this increase in potency observed in the clinic, the dose–response curves for target reduction of three liver-derived proteins that are present in plasma are presented and adding a GalNAc targeting ligand consistently increases potency by 15- to 30-fold compared with the parent PS 2ʹ-MOE PS ASO. The increased potency supports 15- to 30-fold lower dosing, which results in reduction to nearly zero events that could affect compliance such as ISRs and fever and chills that occur in a few patients with 200 to 300 mg/week doses of the parent drugs. Moreover, the GalNAc-conjugated PS 2ʹ-MOE ASOs can be administered monthly to quarterly with essentially no safety or tolerability issues.
      A comparison of the performance of Factor XIRx, a well-behaved PS MOE ASO that targets a critical hepatocyte-derived clotting factor in the intrinsic coagulation pathway to the behavior of Factor XI-LRx, the same PS MOE ASO, is another example of the power of GalNAc conjugation. The parent PS 2ʹ-MOE ASO has completed several phase 2 studies and is poised to enter phase 3 trials. In a study of 300 patients who underwent voluntary knee replacement, three dose levels of the ASO were studied and compared with the standard of care, heparin and the 300 mg/week dose showed a sevenfold lower incidence of deep vein thrombosis with no significant increase in bleeding (
      • Buller H.R.
      • Gailani D.
      • Weitz J.I.
      Factor XI antisense oligonucleotide for venous thrombosis.
      ,
      • Bethune C.
      • Morgan E.
      • Schulz D.
      • Yu R.
      • Jung S.W.
      • Geary R.S.
      • Bhanot S.
      IONIS-FXI-LRx, a FXI GalNAc conjugated antisense drug, produces potent and sustained reductionin FXI activity in normal volunteers.
      ). A similar profile has been observed in patients with end-stage renal disease. The GalNAc version of the Factor XI PS MOE ASO has advanced to phase 2 studies. These agents represent a substantial advance in the management of thromboembolic disorders, and the discovery of these agents is representative of the power of antisense technology. In mice, multiple liver-derived clotting factors were reduced simultaneously with PS MOE ASOs without evidence of nonspecificity and no adverse events. All of the ASOs were then evaluated in various models of thrombosis, and the Factor XI ASO displayed a clearly more attractive profile than other factors (
      • Zhang H.
      • Lowenberg E.C.
      • Crosby J.R.
      • MacLeod A.R.
      • Zhao C.
      • Gao D.
      • Black C.
      • Revenko A.S.
      • Meijers J.C.
      • Stroes E.S.
      • Levi M.
      • Monia B.P.
      Inhibition of the intrinsic coagulation pathway factor XI by antisense oligonucleotides: A novel antithrombotic strategy with lowered bleeding risk.
      ).

      PS cEt GalNAc ASOs

      Recall that PS cEt and PS MOE ASOs share common pharmacokinetic properties, but PS cEt ASOs are approximately ten times more potent. Not surprisingly, conjugating a GalNAc moiety increases the potency for hepatocyte targets by 15- to 30-fold resulting in the most potent chemical class of PS ASOs identified to date. The ED50 of PS cEt ASOs is 1 to 3 mg/week, meaning the total annual dose for a patient is 50 to 150 mg. Obviously then, given that the elimination half-life from tissues is 3 to 4 weeks as discussed previously and the excellent safety profile of the current cET PS Gal-nac ASOs, monthly, quarterly, or semiannual dosing SQ is feasible (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ). Though the total clinical experience is less than with PS 2ʹ-MOE GalNAc ASOs, several hundred humans have been treated systemically (SQ) and by aerosol administration. To date, no ISRs and no drug-related adverse events have been observed.
      Given the oral bioavailability and oral activity demonstrated by 2ʹ-MOE mipomersen (a PS 2ʹ- MOE ASO) in several species including humans (
      • Hardee G.E.
      • Tillman L.G.
      • Geary R.S.
      Routes and formulations for delivery of antisense oligonucleotides.
      ,
      • Yu R.Z.
      • Kulkarni K.H.
      • Tillman L.
      • Prakash T.P.
      • Seth P.
      • Swayze E.
      • Geary R.
      • Monia B.P.
      • Henry S.P.
      • Wang Y.
      Investigation of oral delivery of GalNac3 conjugated antisense oligonucleotides in rats.
      ), the enormous increase in potency achieved by combining the cEt and GalNAc modifications suggests that a PS 2ʹ cEt GalNAc ASO might be bioavailable at doses and formulations that are commercially attractive. Indeed, that appears to be the case. Formulated essentially as mipomersen, PS cEt GalNAc ASOs to several different targets expressed in hepatocytes were approximately 7 to 10% bioavailable and reduced their specific mRNAs in several animal species (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Tillman L.G.
      • Geary R.S.
      • Hardee G.E.
      Oral delivery of antisense oligonucleotides in man.
      ,
      • Fox A.H.
      • Lam Y.W.
      • Leung A.K.
      • Lyon C.E.
      • Andersen J.
      • Mann M.
      • Lamond A.I.
      Paraspeckles: A novel nuclear domain.
      ,
      • Bennett C.F.
      Therapeutic antisense oligonucleotides are coming of age.
      ). One of these ASOs is currently undergoing clinical testing of both the SQ and oral forms of the drug (
      • Yu R.Z.
      • Kulkarni K.H.
      • Tillman L.
      • Prakash T.P.
      • Seth P.
      • Swayze E.
      • Geary R.
      • Monia B.P.
      • Henry S.P.
      • Wang Y.
      Investigation of oral delivery of GalNac3 conjugated antisense oligonucleotides in rats.
      ,
      • Judge D.P.
      • Kristen A.V.
      • Grogan M.
      • Maurer M.S.
      • Falk R.H.
      • Hanna M.
      • Gillmore J.
      • Garg P.
      • Vaishnaw A.K.
      • Harrop J.
      • Powell C.
      • Karsten V.
      • Zhang X.
      • Sweetser M.T.
      • Vest J.
      • et al.
      Phase 3 multicenter study of revusiran in patients with hereditary transthyretin-mediated (hATTR) amyloidosis with cardiomyopathy (ENDEAVOUR).
      ).
      That the intact PS cEt GalNAc ASO was found in the liver in all species studied is an interesting observation because the GalNAc moiety is coupled to the PS ASO via a cleavable linker suggesting that a clinically meaningful fraction of the intact drug is absorbed from the gut. The observation that significant pharmacological activity was observed in humans at a dose that can be administered in a single capsule or tablet and at this dose suggests that commercially attractive oral administration of this chemical class of PS ASOs is feasible, which would be a major advance in the technology that could expand the breadth of utility of antisense drugs yet again (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Tillman L.G.
      • Geary R.S.
      • Hardee G.E.
      Oral delivery of antisense oligonucleotides in man.
      ,
      • Yu R.Z.
      • Kulkarni K.H.
      • Tillman L.
      • Prakash T.P.
      • Seth P.
      • Swayze E.
      • Geary R.
      • Monia B.P.
      • Henry S.P.
      • Wang Y.
      Investigation of oral delivery of GalNac3 conjugated antisense oligonucleotides in rats.
      ).

      PS 2ʹ F ASOs and siRNAs

      A fluoro (F) substitution at the 2ʹ deoxy position is another modification that has been studied. It is rarely used in PS ASOs, but is still frequently substituted at a number of nucleotide positions in both the sense and antisense strands of siRNAs and is present at some level in almost all GalNAc-conjugated siRNAs (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Setten R.L.
      • Rossi J.J.
      • Han S.P.
      The current state and future directions of RNAi-based therapeutics.
      ,
      • Tang X.
      • Zhang S.
      • Fu R.
      • Zhang L.
      • Huang K.
      • Peng H.
      • Dai L.
      • Chen Q.
      Therapeutic prospects of mRNA-based gene therapy for glioblastoma.
      ,
      • Fougerolles A.R.D.
      • Maraganore J.M.
      Discovery and development of RNAi therapeutics.
      ,
      • Anguela X.M.
      • High K.A.
      Entering the modern era of gene therapy.
      ). It is, of course, electron withdrawing and hydrophobic. It increases the Tm per nucleotide by approximately 2º, but does not impart any resistance to degradation by nucleases (
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ). Several properties of 2ʹ F nucleosides and 2ʹ F containing oligonucleotides have generated concern. The ultimate metabolic product of any oligonucleotide is a nucleotide, which may be further metabolized by phosphatases. 2ʹ F nucleotides and nucleosides are substrates for the enzymes of the nucleotide salvage pathway and therefore are incorporated into DNA and RNA and inhibitory of both DNA and RNA polymerases (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Jordheim L.P.
      • Durantel D.
      • Zoulim F.
      • Dumontet C.
      Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases.
      ). 2ʹ F nucleoside analogs have been studied extensively as anticancer and antiviral agents and were found to be quite toxic. Fialuridine (FIAU), a fluoro-containing nucleoside analog that showed promise initially, but in humans resulted in severe hepatotoxicity, mitochondrial dysfunction, metabolic acidosis, and death (
      • Fox A.H.
      • Lam Y.W.
      • Leung A.K.
      • Lyon C.E.
      • Andersen J.
      • Mann M.
      • Lamond A.I.
      Paraspeckles: A novel nuclear domain.
      ,
      • McKenzie R.
      • Fried M.W.
      • Sallie R.
      • Conjeevaram H.
      • Di Bisceglie A.M.
      • Park Y.
      • Savarese B.
      • Kleiner D.
      • Tsokos M.
      • Luciano C.
      • Pruett T.
      • Stotka J.L.
      • Straus S.E.
      • Hoofnagle J.H.
      Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B.
      ). More recently, PS and 2ʹ F containing ASOs were shown to be hepatotoxic due to binding avidly to and causing the cellular degradation of several paraspeckle proteins including P54nrb (Non-POU domain-containing octamer-binding protein) and PSF (splicing factor proline and glutamine rich), which resulted in P53 activation, necrosis, and apoptosis in vitro and in mouse liver (
      • Shen W.
      • Liang X.H.
      • Sun H.
      • Crooke S.T.
      2'-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF.
      ,
      • Shen W.
      • De Hoyos C.L.
      • Sun H.
      • Vickers T.A.
      • Liang X.H.
      • Crooke S.T.
      Acute hepatotoxicity of 2' fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins.
      ). This issue is an important concern because most currently used siRNAs contain 2ʹ F nucleotides, and several have been terminated from development, including revusiran. Revusiran is GalNAc-conjugated siRNA that contains 2ʹ F, 2ʹ methoxy and PS moieties. It is designed to treat patients with TTR amyloidosis. A phase 3 study of patients with TTR cardiomyopathy was discontinued because treated patients experienced severe hepatotoxicity, metabolic acidosis, and death significantly greater than the placebo group. The concern is exacerbated by the fact that details of all the adverse events including time courses for affected patients, as well as mechanistic studies in those patients have never been published (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Judge D.P.
      • Kristen A.V.
      • Grogan M.
      • Maurer M.S.
      • Falk R.H.
      • Hanna M.
      • Gillmore J.
      • Garg P.
      • Vaishnaw A.K.
      • Harrop J.
      • Powell C.
      • Karsten V.
      • Zhang X.
      • Sweetser M.T.
      • Vest J.
      • et al.
      Phase 3 multicenter study of revusiran in patients with hereditary transthyretin-mediated (hATTR) amyloidosis with cardiomyopathy (ENDEAVOUR).
      ,
      • Janas M.M.
      • Zlatev I.
      • Liu J.
      • Jiang Y.
      • Barros S.A.
      • Sutherland J.E.
      • Davis W.P.
      • Liu J.
      • Brown C.R.
      • Liu X.
      • Schlegel M.K.
      • Blair L.
      • Zhang X.
      • Das B.
      • Tran C.
      • et al.
      Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.
      ). Rather, a superficial report of the study that suggested a minor difference in the age of patients in the revusiran arm of the study might be accountable. However, in a later published mechanistic study in animals, the authors concluded that most likely cause of the adverse events was off-target effects due to the promiscuity of AGO2 (
      • Judge D.P.
      • Kristen A.V.
      • Grogan M.
      • Maurer M.S.
      • Falk R.H.
      • Hanna M.
      • Gillmore J.
      • Garg P.
      • Vaishnaw A.K.
      • Harrop J.
      • Powell C.
      • Karsten V.
      • Zhang X.
      • Sweetser M.T.
      • Vest J.
      • et al.
      Phase 3 multicenter study of revusiran in patients with hereditary transthyretin-mediated (hATTR) amyloidosis with cardiomyopathy (ENDEAVOUR).
      ,
      • Janas M.M.
      • Zlatev I.
      • Liu J.
      • Jiang Y.
      • Barros S.A.
      • Sutherland J.E.
      • Davis W.P.
      • Liu J.
      • Brown C.R.
      • Liu X.
      • Schlegel M.K.
      • Blair L.
      • Zhang X.
      • Das B.
      • Tran C.
      • et al.
      Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.
      ).

      PS 2ʹ LNA ASOs

      As discussed earlier, PS 2ʹ LNA ASOs are more toxic in in vitro assays and mouse and NHPs than other 2ʹ modified PS drugs. In the clinic, several different PS 2ʹ LNA ASOs were shown to be toxic with severe thrombocytopenia, severe liver toxicity, and nephrotoxicity being observed. Thus, no clinical trials with ASOs of this chemical class are currently underway. However, efforts to select safe LNA-modified ASOs and to ameliorate the toxicities continue.

      PS ASO interactions with proteins

      Several years ago, the conceptual basis and practice of antisense technology underwent a tectonic shift occasioned by cumulative data that supported the conclusion that the fate of PS ASOs in biological systems is defined by proteins (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ). This means that if a PS ASO is found at a site in a biological system, a protein or proteins are responsible for the PS ASO being there. It also means that all known post-RNA-binding mechanisms are mediated by proteins and often can be influenced by other proteins. Finally, it means that the major mechanism that explains the toxicities of some PS ASOs, irrespective of 2ʹ modification, is driven by unique interactions with paraspeckle proteins and RNase H1 that cause the formation of a unique PS ASO ribonucleoproteins (RNPs) that relocalize to the nucleolus causing nucleolar toxicity and apoptosis (
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Shen W.
      • De Hoyos C.L.
      • Migawa M.T.
      • Vickers T.A.
      • Sun H.
      • Low A.
      • Bell 3rd, T.A.
      • Rahdar M.
      • Mukhopadhyay S.
      • Hart C.E.
      • Bell M.
      • Riney S.
      • Murray S.F.
      • Greenlee S.
      • Crooke R.M.
      • et al.
      Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.
      ,
      • Lima W.
      • Wu H.
      • Crooke S.T.
      The RNase H mechanism.
      ,
      • Vickers T.A.
      • Crooke S.T.
      Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms.
      ). Two lines of research, targeted delivery and studies on molecular mechanisms, drove the advances in understanding PS ASO behavior and the shift in focus. The data supporting this important conclusion is substantial, continues to accumulate, and will be discussed as part of the discussions of pharmacokinetics, cell uptake and distribution, mechanisms of pharmacodynamics, and toxicities.
      For more than 2 decades, the primary focus was to understand the specifics of PS ASO interactions with RNA. In effect, the field was focused on the nucleic acid language and gratifyingly, today we have a solid working knowledge of the factors that determine the ability of a specific PS ASO of a specific chemical class to bind specifically to its cognate “receptor” sequence (Table 2).We also have a large chemical toolbox (
      • Crooke S.T.
      • Witztum J.L.
      • Bennett C.F.
      • Baker B.F.
      RNA-targeted therapeutics.
      ,
      • Crooke S.T.
      • Liang X.-H.
      • Crooke R.M.
      • Baker B.F.
      • Geary R.S.
      Antisense drug discovery and development technology considered in a pharmacological context.
      ,
      • Crooke S.T.
      • Baker B.F.
      • Crooke R.M.
      • Liang X.-H.
      Antisense technology: An overview and prospectus.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Swayze E.E.
      • Bhat B.
      The medicinal chemistry of oligonucleotides.
      ,
      • Crooke S.T.
      • Vickers T.A.
      • Lima W.F.
      • Wu H.-J.
      Mechanisms of antisense drug action, an introduction.
      ) such that the primary focus today is to understand the effects that posttranscriptional modifications may have on PS ASO–RNA interactions. However, given the obvious importance of PS ASO interactions with proteins, during the last several years substantial efforts to understand the chemistry of PS ASO–protein interactions have rapidly yielded important and useful insights (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ).
      Table 2Structure activity relationships of PS ASO-RNA and PS-ASO protein interactions
      PS ASOProtein
      ✓ Phosphorothioates✓ Domains (for key proteins)
       o Number✓ Structures
       o Placement✓ Charge
      ✓ Charge✓ Hydrophobicity
      ✓ 2′ modifications▪ Modifications
       o Hydrophobicity ▪ Acylation
       o Number ▪ Mesylation
       o Orientation (5′ or 3′) ▪ Phosphorylation
      ▪ Sequence ▪ Glycosylation
      ✓ Base modifications ▪ Lipidation
      ▪ Pendant groups (conjugates) ▪ Ubiquitinylation
      ✓ More solid knowledge.
      ▪ Less knowledge.
      Several high-yield efficient assays contributed to the rapid progress. A biotin pull-down assay with PS ASOs as bait and a variety of PS ASOs used as competitors has supported rapid identification of the total proteins that bind a specific PS ASO, identifying how different cellular conditions affect PS ASO–protein interactions and preliminary structure–activity relationships (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.
      ,
      • Liang X.H.
      • Sun H.
      • Shen W.
      • Crooke S.T.
      Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages.
      ,
      • Liang X.H.
      • Shen W.
      • Sun H.
      • Prakash T.P.
      • Crooke S.T.
      TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells.
      ). A modification of the NanoBRET assay that was originally designed to measure protein interactions that supports the evaluation of PS ASO–protein interactions has been particularly instrumental because it is rapid, works well with isolated proteins or mixtures, does not require a protein-specific antibody (antibodies to nano-luciferase can be used), supports direct or competitive binding assays, is rapid, reproducible, and consistent with other binding assays. This procedure even supports estimating the region in a protein that binds and the orientation of the oligonucleotide (
      • Crooke S.T.
      • Vickers T.A.
      • Liang X.H.
      Phosphorothioate modified oligonucleotide-protein interactions.