evropský sociální fond v ČR EVROPSKÁ UNIE MINISTERSTVO ŠKOLSTVÍ MLÁDEŽE A TĚLOVÝCHOVY OP Vzdělávání pro konkurenceschopnost INVESTICE DO ROZVOJE VZDĚLÁVÁNÍ Antisense oligonucleotides © Oldřich Farsa 2020 1 Antisense RNA • Expression vector that produces a transcript, which is complementary to a known transcript • Expression vector is introduced into a host cell by transfection and blocks translation of target mRNA 2 Antisense RNA AS cDNA pa J Transcription Antisense RNA p 1 2 pa t A Transcription Primary transcript RNA processing mRNA mRNA-anti sense RNA complex No translation Therapeutic Antisense RNA • Insulin-like growth factor 1 • prevalent in malignant glioma (common brain tumour) as well as prostate carcinomas • Rat prostate carcinoma cells transfected with antisense receptor cDNA • Mouse injected with transfected cells • small or no tumours compared to control rats treated with non-transfected carcinoma cells Why Antisense Oligonucleotides? • Large antisense RNA is readily subjected to degradation • Human cells specifically target dsRNAfor turnover by nucleases • DNA oligonucleotides more stable, more readily delivered to target cells • Directed to 5' or 3' ends of mRNAs, intron-exon boundaries, naturally ds regions Antisense Oligonucleotides Antisense oligonucleotide 1 2 Transcription 1 2 Primary transcript RNA processing t t mRNA-antisense RNA complex _J_ No translation Antisense Oligonucleotides - „mode of action" DNA 'is mKINA Protein Oligo rii.....rill RN H Possibilities of RNA-triplex forming by action of antisense-oligos (f \. N T O Hoogsleen base pairing \ T O Watson-Crick base pairing R—N C (+)/)— )-#> O H ■■ N Hoogsteen base pairing N 1ST H Fig, 6,11 Watson-Crick and Hoogsteen base pairing. Possibilities of RNA-triplex forming by action of antisense-oligos (continued) reverse Hoogsteen base pairing Watson-Crick base pairing reverse Hoogsteen base pairing Watson-Crick base pairing reverse Hoogsteen base pairing 00 \ R N I R Watson-Crick base pairing Fig. 6.12 Possible triple helix motifs. Optimizing Antisense Oligos • Natural oligos (A) susceptible to cellular nucleases •Alterations improve nuclease resistance A 3'' 0 1 0=P—OH I base \^Uy>base B O base 0 1 0=P—SH I 0. .0 >X1 base • Replacement of free oxygen with sulfur in phosphodiester bond (B) particularly effective • Replacement of deoxyribose with morpholine ring together with substitution of hydroxyl group in phosphate moiety (D) with dimethylamine led also to active compounds D O base NH I 0=P—OH i. .0 base base Ö-CH3 H,C (3*)N \ N—P—O / HaC O Phosphorothioate Antisense Oligos (B) • Water-soluble in form of polysodium salts • Complex with target mRNA activates RNase H • Some therapeutics in clinical trials • eg. against cytomegalovirus infection of retinas in patients with AIDS • Decreased mRNA binding, increased nonspecific protein binding wrt native oligos • Binding of oligos preven inappropriate splice activity Native oligos: Splice Site Correction A • (3-thalassemia: Mutations in hemoglobin chains lead to reduced oxygen binding (anemia) 1 2 k 3 RNA processing b B2 1 2 3 RNA processing t Oligos' Delivery Extremely hydrophilic Exactly tissue-targeted ~ i-/\aoiiy uoouc-iaiycicu O ^ ^ O delivery needed Q^^^^Stf sP • Liposome plus cell-specific binding proteins could allow targeted, efficient delivery of appropriate doses of therapeutic antisense oligonucleotides Hydrophilic Compounds Used fomivirsen •21 deoxyribonukleotides, phosphorothioate •docosasodium salt •sequence 5'-G-C-G-T-T-T-G-C-T-C-T-T-C-T-T-C-T-T-G-C-G-3' • cytomegalovirus replication inhibitor: complementary to RNAof IE2 ^immediate early region 2") of HCMV, inhibits IE2 protein production and thus virus replication •treatment of CMV retinitis in patients with AIDS •Vitravene7l intravitreal inj. (approved in USAcca 1998 - 2001) Compounds in Clinical Trials Phosphorothioate oligos alicaforsen - therapeutic of ulcerative colitis, inh. of synt. ICAM (intercellular adhesion molecule afovirsen - antiiotic & antineoplastic - inhibitor of human papillomavirus replication S D Ii 11 11 aprinocarsen, Affinitak7i- antineoplastic - inhibitor of protein kinase C r non small cells lung & breast cancer oblimersen, Genasensei antineoplastic ■ targeted to gene of Bcl2 anti-apoptic protein - various tumours trabedersen, AP-12009 - antineoplastic ■ inhibitor of overexpression of Transforming Growth Factor \ (TGF \) - brain cancers (glome, astrocytome 1 Compounds in Clinical Trials Intercellular Adhesion Molecule (ICAM) Synthesis Inhibitors Ulcerative Colitis = Inflammatory Bowel Disease (IBD) = chronic relapsing inflammatory disease of the mucose layer of the intestine •idiopathic = ethiology is unknown •pathogenesis is believed to be multifactorial and to include genetic, environmental and immunologic factors •chronic inflammation is manifested namely due to dysregulation of the adaptive immunity system, which leads to a change of tolerance to intestinal bacteria and to an anomalous response to the normal luminal microflora O immunologic imbalance O * production of inflammatory cytokines and adhesion molecules (ICAM, Mad CAM), * activation of polymorphonuclear monocytes (PMN); their migration into the intestine and interaction with the epithelium influences epithlium functions from the barrier one to electrolytes management INTESTINAL LliMEfr t~|NJ1hcliill L'dlü PMN Eti'F VISU LZL MAB ALICAFOR5EN ; Lvnphocyf* INFLIXIMAB II ■ 11 ■ v-vr\i i/.y mau M LN-Ü2 t DP?-1 [.AM IM A l'kOPRIA BA£[LIXIMAB DAC LIZ LI IM AB Mechanism of origin of IBD, biomolecules engage in it and therapeutic targets of selected biodrugs ICAM-1 intercelullar adhesion molecule 1 MadCAM mucous address adhesion molecule I FN interferon IL interleukin EGF epidermal growth factor PMN - polymorphonuclear monocyte o. H HO. Alicaforsen syn. ISIS 2302 A H2VN o ^V^. Vn^ •oNgodeoxyribonucleotide modified by N O S o^o oA H2N ^=^0 thiophosphoric acid (phosphorothioate) O S O' o V H2Nv.N. •20 bases ^N "binds (hybridizes) to the non-translated 3'-region o" of human ICAM-1 mKNA O activation of r in °PS (A N^>N OS p\ nuclease RNasy-H O cleavage of the s nh2 heterodimer O attenuation of ICAM synthesis °;p. H3\\>4H H 6^3 . nA-N n H °/ O^ N^>N b s cA n^Vn •phase 3 clinical tests o^p°—vs H o h °;p'^ oA H2N o s VN' O' ^ ■-. o^ o O" o Compounds in clinical trials: antiviral agents 8 afovirsen, syn. ISIS 8741 - free „acid"; ISIS 2105 - nonadecasodium salt •Sequence: ttgcttccat cttcctcgtc •Modification: residues of phosphoric acid substituted with those of thiophosphoric acid •Usage: treatment of human papillomavirus infections (HPV; manifestations of the infection: warts of female genitals - cervical cancer) •mode of action: binding to viral mRNA T _ Effect of ISIS 2105 and ISIS 3925 (20 Kj0.. residue phosphorothioate oligonucleotide designed to be complementary to influenza A virus) to HPV DNA replication. 24 h after infection by electroporation, cells of the line SCC-4 were treated with increasing doses of either ISIS 2105 or ISIS 3925. Cells were „harvested" 24 h after electroporation and their HPV DNA content was determined. Data are plotted in % of untreated control experiment; mean of 3 assays. !50 75 100 125 15Ü T7S 20Ö 225 Olijonuc:öütide (nJul) -ISIS i-ns ,--- 13153035 Compounds in clinical trials: antiviral agents miravirsen, SPC3649 •RNA, (P-thio)((2,-0,4,-C-methylene)m5C-dC-(2,-0,4,-C-methylene)A-dT^^ methylene)G-(2,-0,4,-C-methylene)m5U-dC-dA-(2,-0,4,-C-methylene methylene)m5C-dT-(2,-0,4,-C-methylene)m5C-(2,-0,4,-C-methylene)m sodium salt (1:14) •16 bases, phosphorothioate groups, 8 methylene bridges between 2'-0 and 4'-C atoms of ribose •microRNAs are short (21-23 nucleotide) non-coding regulatory RNAs that influence gene expression at a post-transcriptional level • microRNA-122 (miR-122) is a major regulatory RNA in liver that fine-tunes the expression of over 100 cellular genes and enhances hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5' untranslated region (5' UTR) of the viral RNA •Jocked nucleic acid" SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo •phase 1 - 2 clinical tests against HCV infection have been finished 2 Compounds in clinical trials: antitumour agents Proteinkinase inhibitors •proteikinases C: serine-threonine kinases taking part in cell signal transduction (phosphorylation of signal molecules); their altered expression is linked with cancerogenesis aprinocarsen, syn. ISIS 3521, LY900003, CGP 64128A Affinitak7i •deoxyribonucleotide, 20 bases, phosforothioate •A-C-T-T-T-G-A-G-T-G-G-T-C-G-C-T-C-T-T-G •phase 2 clinical tests against non-small cells lung cancer (inoperable or metastasizing) alone or combined with carboplatin, gemcitabin or paclitaxel; also against breast cancer treated previously by an other drug have been finished •phase 3 against lung cancer: addition of aprinocarsen to gemcitabin or carboplatin has neither prolonged patients survival nor increased treatment efectiveness evaluated by other measures 2 Compounds in Clinical Trials Morpholine oligos eteplirsen, AVI-4658, Exondys 51 ® •treatment of Duchenne muscular dystrophy (DMD) •DMD: a fatal muscle degenerative disorder, caused by misreading and bad translation of DMD (dystrophin) gene to dystrophin peptide due to mutations of this gene •1 per 3500 newborn boys is affected •sequence CTCCAACATCAAGGAAGATGGCATTTCTAG; 30 bases; Mr = 10369.83 •targeted to 51. exone in dystrophine mRNA •enables partial restoration of reading and translation of DMD gene by ..patching" of its bad splicing O production of truncated, but functional chain of dystrophine •applied locally by i.m. injection in clinical trials •therapeutically given i.v., 30 mg/kg, in 35 - 60 min long infusion once weakely •indication: DMD treatment in u patients with confirmed DMD gene mutation, where „skiping" of exone 51 is available FJg we l: D el eti ons and pred Ictcd, r esu Its of ex on s ki pp ing i n the pat it n ts who w ere it ud.it d (A) Prc-mRIMA transcripts and dystrophin protein products from full length DMDr in patients with Duchennc muscular dystrophy, and predicted protein sequences after exon skipping. (II The normal dystrophin gene produces the full length dystrophin product. (II) Patients land 2 had a deletion in exon 50 that disrupts the open leading frame, leading to a truncated and unstable dystrophin. (Ill) Skipping of exon 51 restores the reading frame, producing a truncated but functional dystrophin that lacks exons 50 and 51. (IV) Patient? is missing exons 49 and 50. (V) Patients^ and 4 are missing cxons48-5Q. (VI) Patients 5 and 6 are missing exons45-50. All thetmncated ctystraphins produced after skipping of exon 51 aie missing the hinged region and some of the rod domain but have been assodatcd with the milder EM D phenotype**1 (Ei) Structure of the phosphorodiamidate morpholino modification of the antisense oligomer. Compounds in Clinical Trials Morpholine oligos radavirsen, AVI-7100 •phosphorodiamidate morpholino antisense oligomer with positive charges on selected subunits; 20 nucleotide bases HO n= 1 -19 B(1-20): C-G-G-T-T-A-G-A-A-G-A-C-T-C-A-T-C-T-T-T R(1-3) = R(5-11) = R(13-16) = R(1 &j = R(19) = -N(CH5)2 /—\ R(4) = R(12) = R[17)= HN - •intended for influenza A •specifically targets viral messenger RNA sequences •phase 1 clinical tests to characterize the safety, tolerability and pharmacokinetics of escalating single-administration doses of AVI-7100 in healthy human subjects. 2 Compounds in clinical trials Inhibitors of proteinkinase •proteikinases C: serine-threonine kinases involved in cell signal transduction ( =signal molecules phosphorylation); their altered expression is linked with cancerogenesis aprinocarsen, syn. ISIS 3521, LY900003, CGP 64128A Affinitak7i •deoxyribonucleotide, 20 nucleotides, phosphorthioate •A-C-T-T-T-G-A-G-T-G-G-T-C-G-C-T-C-T-T-G •2. phase clinical tests against non-small cell lung cancer and melanoma (inoperable or methasthatic, alone or with carboplatin, gemcitabin or paclitaxel; also for previously treated breast cancer finished •3. phase agains lung cancer: addition of aprinocarsen to gemcitabin or carboplatin did not prolong pacients survival nor increased treatment efficacy rated by other criteria Compounds in clinical trials: antincancer drugs Oncogene Bcl-2 antagonist •Bcl-2: antiapoptic protein; its dominancy over structuraly related proapoptic Bax presents a bad response of tumors to common anticancer therapies and bad prognosis oblimersen, G3139, augmerosen, Genasense-^ •deoxyribonucleotide, 18 nucleotides, phosphorthioate, heptadecasodium salt •T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T •complementary to first six codones of human Bcl-2 mRNA •administered by podání i. v. infusion •in 1st - 3rd phases clinical trials agaist various types of cancer; efficient; rel. low toxicity