2 Mechanisms of antisense action on target genes Mechanisms of antisense action on target genes. Several mechanisms of antisense action have been determined for antisense drugs (shown in green). These include inhibition of transcription (a), inhibition of splicing and inhibition of mRNA maturation by prevention of 5'-cap formation and of polyadenylation (b), and inhibition of ribosomal readthrough (c). Most importantly, antisense drugs hybridized with target RNA have been shown to serve as substrates for RNase H enzymes (shown in red; b,d) that cleave the RNA in DNA–RNA hybrids or RNA–RNA hybrids (as in RNAi) and decrease mRNA levels of the target gene. So, there is a growing repertoire of potential mechanisms of
3 Triple-helix formation at the nucleotide level Triple-helix formation at the nucleotide level. Shows the formation of Watson–Crick (red) and Hoogsteen bonds (black) between duplex pairs and the third strand (the arrow points to a single base of the third strand). Blue, guanine residue (purine); pink, cytosine residue (pyrimidine).
4 Mechanisms of antisense action on target genes Mechanisms of antisense action on target genes. Several mechanisms of antisense action have been determined for antisense drugs (shown in green). These include inhibition of transcription (a), inhibition of splicing and inhibition of mRNA maturation by prevention of 5'-cap formation and of polyadenylation (b), and inhibition of ribosomal readthrough (c). Most importantly, antisense drugs hybridized with target RNA have been shown to serve as substrates for RNase H enzymes (shown in red; b,d) that cleave the RNA in DNA–RNA hybrids or RNA–RNA hybrids (as in RNAi) and decrease mRNA levels of the target gene. So, there is a growing repertoire of potential mechanisms of
5 Mechanisms of nucleic-acid-based approaches for gene silencing: antisense compounds. Two mechanisms by which antisense compounds sequence-specifically alter gene expression. Oligodeoxyribonucleic acids (ODNs) can be introduced into the cell through experimental manipulation. The antisense molecules can hybridize to either mRNA or pre-mRNA. The RNA strand of DNA–RNA duplexes is degraded by RNase H. Certain chemically modified antisense molecules complexed with RNA are not recognized by RNase H. These types of compound can be used to inhibit translation of mRNAs or inhibit or alter splicing pathways of pre-mRNAs.
8 ISTRUZIONI PER L’USO DEGLI OLIGONUCLEOTIDI ANTISENSO Utilizzare controlli appropriatiVerificare l’inibizione dell’espressione del bersaglioUtilizzare le minime concentrazioni necessarie a inibire il bersaglioNon utilizzare oligonucleotidi contenenti 4 residui di guanina contiguiNon utilizzare oligonucleotidi contenenti un motivo CpG non modificato (negli esperimenti con gli animali)
9 PROBLEMI LEGATI ALL’USO DEGLI OLIGONUCLEOTIDI ANTISENSO Stabilità biologicaTargeting cellulareUptake cellulareEfflusso dagli endosomi
10 Mechanisms of nucleic-acid-based approaches for gene silencing: ribozymes. General mechanism by which ribozymes silence gene expression. Ribozymes can be produced within the cell through transcription or can be directly introduced into the cell through experimental manipulation. For the hammerhead ribozyme, two arms are used to direct the catalytic centre to target the hydrolysis of the phosphodiester backbone of the mRNA.
11 APTAMERISequenze oligonucleotidiche (ssDNA o ssRNA) o peptidiche relativamente brevi (12-30 nucleotidi o aminoacidi)In vivo assumono strutture tridimensionali specifiche in grado di legarsi con elevata affinità (range nM/pM) alle proteine bersaglio
12 SELEX systematic evolution of ligands by exponential enrichment Scheme of systematic evolution of ligands by exponential enrichment (SELEX). A library of DNA or RNA molecules is incubated with the protein target, and those that bind to it are separated from the rest. The sequences with affinity for the target are subsequently amplified to generate a pool of molecules that bind to the protein of interest.
14 APTAMERI vs. ANTICORPI Maggiore stabilità Non immunogenici Ridotta variabilità da un lotto all’altroPiccole dimensioni migliore penetrazioneclearance più rapidapossibilità di modificarne la struttura, p.e. per migliorare la stabilità o alterarne le proprietà PK
15 MACUGEN® (pegaptanib sodio) Primo aptamero approvato, nel dicembre 2004, per l’uso clinico nella degenerazione maculare senile (AMD)ssRNA costituito da 28 nucleotidi, specifico per una delle 6 isoforme di VEGFModificato a livello dello zucchero per aumentare la resistenza alle nucleasiaggiunta covalente di due molecole di PEG (20 kD ca.) per aumentare t½
16 b | Simplified schematic of a process by which RNA aptamers — such as pegaptanib — that bind specifically to protein targets can be prepared. A large library of DNA oligonucleotides are transcribed into RNAs, which are selected for their ability to bind to a target, such as VEGF. Those RNAs that bind best are amplified (by reverse transcription, amplification using the polymerase chain reaction and transcription). Successive selection and amplification cycles result in an exponential increase in the abundance of RNA aptamers that bind to the target.
17 MACUGEN® (pegaptanib sodio) a | Sequence and predicted secondary structure of pegaptanib. 2'-O-methylated purines are shown in red, 2'-fluorine-modified pyrimidines are shown in blue and unmodified ribonucleotides are shown in black. The site of attachment of a 40-kDa polyethylene glycol moiety is shown. b | Interaction between the 55-amino-acid heparin-binding domain of vascular endothelial growth factor (VEGF)165 and pegaptanib. Representation of the previously determined NMR solution structure of the free heparin-binding domain of VEGF165 from Fairbrother et al.45 is shown in grey with disulphide bonds in yellow. The aptamer (blue) is shown as a model based on the secondary structure determined by NMR46, with the helical stem regions in teal. The previously reported interaction between cysteine-137 of VEGF165 (cysteine-27 of the heparin-binding domain) and uridine-14 of the aptamer14 is indicated in red.
18 APTAMERI nella terapia dei tumori GB10: DNA aptamero che si lega alla tenascina Captameri peptidici diretti vs. le regioni di dimerizzazione e di legame al DNA di STAT3AS1411: ssDNA 26-mero ricco di guanosina, forma un complesso con la nucleolina e il fattore modulatore essenziale di NFκB (NEMO)
20 Effetto in vitro di AS1411 IC50 ~1.1 SRB cytotoxicity assay of AS1411 in human lung (A549) and prostate (DU145) cells. AS1411 was incubated with the cells in cell culture medium for 6 d. After 6 d, the medium was removed and replaced with 50% trichloroacetic acid. The cells were washed and incubated with sulforhodamine dye, and absorbance was measured at 564 nm. Points, mean (n = 3). Result is representative of three such experiments.~1.1
21 Effetto in vivo di AS1411In vivo effect of AS1411 on the growth of A549 (A) and SKMES (B) human lung cancer xenografts grown s.c. in nude mice. A, vehicle (PBS) or AS1411 was administered daily (5, 10, and 40 mg/kg, i.v.) for 5 consecutive days. Tumor volumes were measured on day 14 and expressed as tumor volume relative to the volume on the initial day of treatment. Columns, mean (n = 8); bars, SD. B, vehicle ( ), 5 mg/kg ( ), or 10 mg/kg ( ) AS1411 i.p. administration on days 0, 2, and 4 from the time tumors had become established ( 100 mm3).
22 AS1411 Studi tossicologici pre-clinici: nessuna tossicità nel ratto e nel caneStudi clinici (fase I) in NSCLC e carcinomi renali