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Clase oligos 2013 Clase oligos 2013 Presentation Transcript

  • Nucleic Acids Therapeutics: Antisense, Antigene and Aptamers Dr. Luis Marat Alvarez Salas Laboratorio de Terapia Génica Departamento de Genética y Biología Molecular CINVESTAV
  • Nucleic Acids: More than meets the eye CODING Proteins SEQUENCES Nucleic Acids Transcription Expression REGULATORY Replication Inheritance SEQUENCES Recombination Variability Single strand Information FORMS Duplex safe-keeping Triplex Primary STRUCTURE Secondary Catalytic Tertiary Activity
  • Nucleotide Structure
  • Oligodeoxynucleotide Structure NH2 NH2 O N N N N N N O N O N H H H adenine cytosine thymine O O N N CH3 HN H2N N N N H O H guanine uracyl
  • Atomic numbering scheme and definition of torsionangles for a polyribonucleotide chain
  • anti and syn conformational ranges for glycosydicbonds in pyrimidine (left) and purine (right)nucleosides
  • Movements of bases in sequence-dependent structures (tip,inclination, opening, propeller, buckle, twist, roll, tilt, slide, rise, shiftand tilt).
  • Secondary Structures in ssRNA Chastain, M. and Tinoco Jr., I., (1991) Prog. Nucleic Acid Res. Mol. Biol. 41, 131-177.
  • Therapeutic nucleic acids (TNA) modes ofaction•Highly specific•Defined chemical structure•Easy to synthesize•Readily available modifications Antisense ODNs AAAA•Cell uptake Gm7•Diverse administration routes Antigene•Relatively low toxic RZ AAAA•Non infectious Gm7•Scalable to industrial TFO DNA RNAi Aptamerproduction AAAA•Adaptable to current clinical Gm7 siRNA/miRNAprotocols DiPaolo JA and Alvarez-Salas LM. 2004. Exp. Op. Biol. Ther. 4:1251
  • ANTIGENE
  • Triplex DNA: Structural Considerations  Hoogsteen (T-A-T, C+-G-C) . Low pH required for cytosine protonation  Homopurine and homopyrimidines chains  Third strand occupies the major groove (parallel motif) and bind to the purine-rich strand (5’-3’)  Antiparallel strand (bind in opposite orientation to the purine- rich strand)  CT>parallel; GA>antiparallel
  • Triplex DNA Parallel motif (CT) 5’-CCTCTTTCCCCTCTTCCCTC-3’ (GT) 5’-GGTGTTTGGGGTGTTGGGTG-3’Target 5’-tgcctGGAGAAAGGGGAGAAGGGAGccttg-3’ site 3’-acggaCCTCTTTCCCCTCTTCCCTCggaac-5’ (GA) 3’-GGAGAAAGGGGAGAAGGGAG-5’ (GT) 3’-GGTGTTTGGGGTGTTGGGTG-5’
  • ANTISENSE
  • Mechanisms of Antisense Action
  • The sense of antisense Ribosome mRNANon-catalytic Catalytic Ribozyme RNA DNA Truncated Protein RNaseH
  • Properties essential for AS-ODN activityTarget accessibilityNuclease stability Cellular Uptake Specificity for target RNA Hybridization by Watson-Crick pairing Affinity for target RNA RNaseH activation for silencing
  • Secondary structure of target HPV-16 mRNA ∆G=-191.6 ∆G= -191.6 Venturini Alvarez-Salas Alvarez-Salas, et al.(1998) AntiE6 419 Alvarez-Salas, et al. (2002) Venturini, et al. (1999)
  • ODN Chemical Synthesis O B DMTO O B DMTO O Oxidation O 5’ Deprotection (Capping) P O aqueous NC O O O B iodine dichloroacteic or dichloroacteic or O B trichloroacetic acid trichloroacetic acidDMTO O O B O HO PNC O O O O B N N N N O tetrazole O B Coupling DMTO O P NC O N
  • Protect from nuclease attack! ANTISENSE TARGET NUCLEASE
  • ODN chemical modifications 5’-palmitate O B O-CH3 O H O-MOE O P O S O B CH3 O N O H
  • ODN chemical modifications H O 5’-Palmitate N O CH2 13derivative O N CH3 O B O H 2’-O-methylphosphorothioate O B O O CH3 S P O O Phosphodiester O B O P O O 2’-O-methoxyethylphosphorothioate O B O O CH3 O O O S P O O O BPhosphorothioate  S P O O α-nucleoside 2’-O-aminopropylphosphodiester O B O O NH2 O O P O O B H3C P O O O B Locked/bridged nucleic Methylphosphonate  O acids O O O O CH32’-O-Methylribonucleotide O P O O P O O B O B O O Phosphoramidate HN HN OH O P O O P O O B O B N3’->P5’Phosphoramidite  O O O O OH O P O O P O O O
  • More chemical modifications BASE BASE O O O O H N N N N N H H H PNA BASE O O O BASE O N O P O P N Morpholino BASE O O O O O BASE N Hexitol nucleic acids
  • ODN administration routes• I.V.• Intramuscular• Subcutaneous• Oral• Topical• Intracraneal
  • PS-ODN Bio-distribution
  • Elimination of PS-ODNs
  • PS-ODN Protein binding Prolongation of partialOff-target Polyanionic nature thromboplastin time Complement activation Effects Thrombocytopenia Antiviral activity Elevation of Phosphorothioate transaminases Oligodeoxynucleotide Antitumor activity Splenomegaly Antimicrobial activity IL-6 Immune stimulation IL-12 (Cytokine induction) Septic shock TNF-α γ-IFN Chemokines IL-1 Acute phase response (MIP, MCP, RANTES) RE cell chemotaxis
  • Reduced Toxicity in ODNs
  • ODN Combined Chemistry 1st generation 2nd generation 3rd generationO O O O O O B B O B O B What is next? O O O O O P O S P O O B O B H3C P O H3C P O O O O B O B O O O O O O O CH3 O P O S P O H3C P O O P O O O B O O B O O O B O B O O O O O P O O S P O O S P O S P O B B O B O B O O O O O O O O O P O S P O S P O S P O O B O B O B O B O O O O O OStandard ODN O P O S P O O H3C P O O O P O O CH3 O B O B O B O B O O O O O O O O O P O S P O H3C P O H3C P O O O O O
  • AS-ODN Pipeline Pre- On Product (form) Target Lead Indication Partner Phase 1 Phase 2 Phase 3 clinical Market Vitravene™(i) Antiviral CMV retinitis Novartis Affinitak™(p) PKC-α Cancer-NCLC Lilly Alicaforsen (p) ICAM-1 Cronh’s disease Isis Alicaforsen (e) ICAM-1 Ulcerative colitis Isis ISIS14803 (p) Antiviral Hepatitis C Elan ISIS 104838 (p,o) TNF-α Rheumatoid arthritis Isis ISIS 104838 (t) TNF-α Psoriasis Isis ISIS 113715 (p) PTP-1B Diabetes Merck ISIS 301012 (p) ApoB-100 Cardiovascular Isis ISIS 112989 (p) Clusterin Cancer-prostate Isis ATL1102 (p) VLA-4 Multiple sclerosis ATL OGX-011 (p) Survivin Cancer Oncogenex i=intravitreal p=parenteral e=enema t=topical o=oral
  • IM-ODNs
  • IFN-α Immunostimulatory ODNs Cytokines TBK1 IRF-3 cytoplasm TRAF6 TAK1 MAPK DNA Translation CpG-ODN IRAK IM-ODN MyD88 AP-1 TRIF TRAF6 IRF-7 IκBTranscription TLR-9 TLR-3 p50 p65 endosome NF-κB GENE TLR-7/8 PROMOTERS dsRNA ssRNA siRNA MyD88 nucleus IRAK TRAF6
  • CLASS A (ODN2216)Strong IFN-α inductionGPSGPSGPGPGPAPCPGPAPTPCPGPTPCPGPGPSGPSGPSGPSG CLASS B (CPG7909 or PF-3512676 or CPG2006) Strong B cell proliferation and plasmacytoid dendritic cells maturation TPSCPSGPSTPSCPSGPSTPSTPSTPSTPSGPSTPSCPSGPSTPSTPSTPSTPSGPSTPSCPSGPSTPST CLASS C (ODN2395) Combined intermediate effects in IFN-α secretion and B cell proliferation 5’-TPSCPSGPSTPSCPSGPSTPSTPSTPSTPSCPSGPSGPSCPSGPSCPSGPSCPSGPSCPSCPSG-3’ 3´-GPSCPSCPSGPSCPSGPSCPSGPSCPSGPSGPSCPSTPSTPSTPSTPSGPSCPSTPSGPSCPST-5’ NH2 NH2 N N O O O N O N O O O O N O HN O HN O P O O P O O N N H2N O N N H2N O O O CpG O CpR
  • Promot er An ti DNA Vaccines ge cccDNA n or th peutic Immunostimulatory sequences (CpG?)Plasmid Strong promoter era No eukaryotic ori Immunostimulation ge neTe rmi n at or AS-RNA Ribozyme Gene silencing shRNA or modulation Intramer Protein nucleus mRNA AAAA Gene expression G m7
  • The hammerhead ribozyme
  • Structure of the hammerhead ribozyme NUX Hybridization NNNNNNNNNCA NNNNNNNNN A C helix I helix III Cleavage A UG G A C GA G U Structure A U G C helix II Cleavage site G C A G G U
  • The hairpin ribozyme Target Rz Rz Target
  • Structure of the hairpin ribozyme GUC 3 CUUAAGAU NNNN NNNNNNNN 5 A 17 A A9 1 N N AG helix 1 helix 2 N N loop 1 helix 3 N N N N 55 C A N 22 54 53U G 23 52U A 24 A 25 Hybridization loop 4 51 A loop 2 50 U A 26 49 A C 27 Catalysis 48 N N 28 47 N Structure 46 N N29 N N N N Stability helix 4 43C G 32 C G U A 40G C 35 loop 3 G U C U
  • Hairpin ribozyme cleavage H2C A-1 H2C A -1 O O O O :B CLEAVAGE H P H:B O P O -O O + OH LIGATION HO H2C G +1 H2C G +1 O OH O OH
  • Ribozyme Clinical Trials Pipeline
  • DNAzymes
  • Cleavage mechanism H2C A -1 H2C A -1 H O O O O :O H P O P O Mg++ -O OH+ O HO H2C G +1 H2C G +1 O OH O OH
  • Ribozyme Diagnostic Applications Allozyme Halfzyme
  • RNAi
  • RNAi mechanism of action dsRNA DICER siRNA Antisense strand RISC complex Target mRNAsiRNA-mediated cleavage Exonucleases
  • Proposed Mechanism for siRNA Action
  • siRNA Sense strand 5- UU-3 3-UU -5 Guide or antisense strand Seed regionSynthetic siRNA PS linkages 2’-modified sugar 5- UU-3 3-UU -5PS linkages Figure 11
  • siRNA design 5’- G Non-hybridized 3’-UU(TT) 5’G U C A A A A G C C A C U G U G U C C U U 3’3’U U C A G U U U U C G G U G A C A C A G G 5’ 19 nucleotides
  • ANTISENSE vs. RNAi ANTISENSE RNAi• No transfection procedure • No gene silencing withoutrequired transfection• Uptake by active transport • Many cell lines like primary cellsmechanisms not transfectable• Technique easy and fast to • Transfection causes cytotoxicestablish effects• Broad application spectrum in • Difficult to use for long orvitro and in vivo constitutive expressed genes• First antisense drug commercially • Limited to in vitro experimentsavailable (Novartis’ Vitravene™) • Still not yet established as a• Many clinical trials worldwide therapeutic(phase III) • Sophisticated design important,• FDA approval because secondary structure• High specificity and efficacy influences accessibility of mRNAthrough Biognostik’s R.A.D.A.R.® • Profound knowledge about RNAidesign design so far not available
  • siRNA off-target effectsSequence-specific gene expression patterns induced by siRNAs. Red bars: fraction MAPK14remaining protein. Black bars: fraction MAPK14 remaining RNA. (In Jackson, A. L., et al. Nat.Biotechnol. 21: 635-637.)
  • siRNA off-target effects γ-IFN ds dependent Antiviral activity siRNA Gene regulation DNA methylation Toll-like receptors TLR-8, TLR-9 ds independent Immune stimulation IL-6 TNF-α IFN-α
  • APTAMERS
  • The SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure ligand Incubation with ligand ligand-RNA or dsDNA ligand-ssDNA RNA/ssDNA ODN complex Library library Selection Aptamers Amplification Unbound RNA/ssDNA
  • Aptamer Structural Diversity
  • Nucleotide Modifications for SELEX R1 = -I , -Br, -SH O O R2 = -F , -NH2 B O R1 NH O O P O O N O O O R2
  • Unnatural Base Pairs in Aptamers
  • Aptamer Applications• Enzyme inactivation• Protein Function Inhibition• Diagnostics
  • Riboreporter Applications
  • Métodos de diagnóstico
  • Ácidos nucleicos funcionales Ribozima Ácidos DNAzima nucleicos Aptasensor funcionales Aptazima
  • Ácidos nucleicos funcionales Ribozima Ácidos DNAzima nucleicos funcionales
  • DNAzimas Cu+2 Metalización. Li & Sen 1996 DNA ligasa. Cuenoud & Szostak 1995DNAzima que mimetiza la actividad peroxidasa. Travascio et al 2001 Hidrolisis de RNA. Reyes-Gutiérrez & Alvarez-Salas 2009
  • DNAzimas H2O2 Luminol DNAzimaDNA blanco Hemina Kosman and Juskowiak 2001
  • DNAzimas Inmovilización Au ▪ Costoso ▪ Equipo ▪ Capacitación ▪
  • Ácidos nucleicos funcionales Ribozima Ácidos DNAzima nucleicos Aptasensor funcionales
  • AptasensoresÁcido nucleico Aptámero catalítico Blanco
  • Aptámeros
  • Aptasensores Membrana Trombina BSABSA Aptasensor ABTS ABTS+ Zhu et al 2010.
  • Aptasensores• Detección de la proteína Inmovilización ▪ • No se requiere biopsia Tiempo ▪ • Altamente específicos y sensibles Equipo ▪• Bajo costo Capacitación ▪
  • Ácidos nucleicos funcionales Ribozima Ácidos DNAzima nucleicos Aptasensor funcionales Aptazima
  • AptazimaÁcido nucleico Aptámero catalítico Blanco
  • Aptazima Aptámero Aptámero DNAzima Excitación (399nm) Blanco NMM Emisión (614nm) Dúplex DNAzima activada FluorescenciaAptazima/antisentido Soo Oh et al 2010.
  • Aptazima• Altas velocidades de reacción • Estables químicamente • Detección de la proteína • No se requiere biopsia • Fácil modificación • Bajo costo • Fácil producción • Detección en solución • Ilimitada vida de anaquel • Altamente específicos y sensibles • No requiere personal y/o equipo capacitado
  • Quadruplex DNA dR H N N N H N N dR N N H O N H O NH N H H N H N O H N O H N NdR N N H N N N H dR
  • DNAzima PS2.M Luminol (5-Amino-2,3-dihidro-1,4-ftalatodiona) ABTS (ácido 2,2–azino–bis–(3–etillbenzotiazolin–6–sulfonico)Hemina Kosman and Juskowiak 2001.
  • DNAzima PS2.MK+ Modificado de Travascio et al 2001.
  • GENETranscription ANTIGENE FUNCTIONS ANTISENSE siRNA/miRNA TranslationDISEASE FunctionGENE APTAMEREXPRESSION
  • ATTACHMENT PENETRATION HOST UNCOATING FUNCTIONS ANTIGENEAPTAMER Transcription ANTIGENE Translation VIRAL ANTISENSE/siRNA/miRNA REPLICATION LIFE ASSEMBLY CYCLE (MATURATION) APTAMER RELEASE MULTIPLICATION