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Peptide-Conjugated Morpholino Oligomers for Treatment of Spinal Muscular Atrophy

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Peptide-Conjugated Morpholino Oligomers for Treatment of Spinal Muscular Atrophy UNIVERSITÀ DEGLI STUDI DI MILANO DOTTORATO IN MEDICINA MOLECOLARE E TRASLAZIONALE CICLO XXX Anno Accademico 2016/2017 TESI DI DOTTORATO DI RICERCA MED/26 PEPTIDE-CONJUGATED MORPHOLINO OLIGOMERS FOR TREATMENT OF SPINAL MUSCULAR ATROPHY Dottoranda: Agnese RAMIREZ Matricola N°R10958-R21 Tutor: Prof. Giacomo COMI Co-Tutor: Prof.ssa Stefania CORTI Coordinatore del dottorato: Prof. Riccardo GHIDONI Sommario L’Atrofia Muscolare Spinale (SMA) è una patologia neuromuscolare autosomica recessiva che colpisce i motoneuroni, ed è, ad oggi, la prima causa genetica di mortalità infantile. E’ causata da mutazioni in omozigosi del gene Survival Motor Neuron 1 (SMN1), che danno luogo alla produzione di insufficienti livelli della proteina SMN. Da un punto di vista patologico, la SMA è caratterizzata da una perdita progressiva dei motoneuroni (MN) nelle corna anteriori del midollo spinale a cui consegue una perdita delle funzioni muscolari, paralisi e morte prematura. Da quest’ anno è stato approvato il primo farmaco per la SMA, Nusinersen, un oligonucleotide antisenso (ASO). Infatti, una delle possibili strategie che permettono di aumentare i livelli di proteina SMN funzionante è l’uso di ASO in grado di modulare lo splicing dell’mRNA di SMN2, il gene paralogo di SMN1. Attualmente sono utilizzati in trial clinici ASO con diverse strutture chimiche, inclusi gli ASO con una chimica di tipo Morfolino (MO) studiati in questo lavoro. Gli oligomeri di tipo Morfolino sono particolarmente utili per applicazioni in vivo grazie al loro buon profilo di sicurezza ed efficacia. Lo scopo di questo studio è di migliorare l’efficacia del MO e di contribuire a risolvere il problema della sua relativa bassa biodisponibilità tissutale. Infatti gli ASO in generale non riescono a superare la barriera emato-encefalica e devono essere somministrati attraverso un’iniezione relativamente invasiva intratecale nel liquido cefalorachidiano per raggiungere il sistema nervoso centrale. In primo luogo, per migliorare l’efficacia degli oligomeri di tipo MO, abbiamo testato nuove sequenze di MO che si appaiano alla regione identificata come intronic splicing silencer N1 (ISS-N1) di SMN2 sia in vitro che in un modello murino transgenico di SMA (SMA7). L’uptake cellulare e tissutale e i profili farmacologici degli ASO possono essere migliorati in modo significativo attraverso la loro coniugazione con cell- penetrating peptides (CPP), ovvero peptidi che riescono a penetrare nelle membrane cellulari, ma questo approccio non è ancora stato utilizzato nella SMA. Quindi, come seconda fase dello studio, ci siamo proposti di valutare un II approccio terapeutico per la SMA utilizzando sequenze MO coniugate a diversi tipi di CPP. Abbiamo testato tre diversi CPP, Tat, R6 e (RXRRBR)2XB che sono stati associati alla sequenza di MO [HSMN2Ex7D(-10,-34)] da noi già precedentemente validata. Abbiamo somministrato i coniugati ottenuti in modelli murini di SMA ad uno stadio pre-sintomatico e sintomatico della malattia per determinare il loro effetto terapeutico rispetto al MO non coniugato. Abbiamo dimostrato che l’ottimizzazione della sequenza target può migliorare l’effetto della correzione dello splicing attuata da MO e che l’effetto della co- somministrazione di più di una sequenza di MO è maggiore rispetto alla somministrazione di una singola sequenza. Inoltre abbiamo dimostrato che i coniugati CPP-MO sono in grado di aumentare sia i livelli di proteina SMN nel sistema nervoso centrale sia la sopravvivenza media e le performance motorie nel modello murino SMA dopo una somministrazione intracerebroventricolare (ICV) e intravenosa (IV). I nostri risultati hanno prodotto dei dati preliminari in grado di sostenere che la coniugazione degli oligomeri di tipo MO ai CPP può essere una strategia attuabile per aumentare l’uptake cellulare dopo una somministrazione intratecale e per veicolare il MO nel sistema nervoso centrale dopo una somministrazione sistemica non invasiva. I risultati ottenuti saranno utilizzati per la selezione del CPP più efficiente che sarà studiato ulteriormente con analisi più approfondite e sarà un punto di partenza per lo sviluppo di futuri trial clinici. III Abstract Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease and the first known genetic cause of infant mortality. It is caused by the homozygous mutations in the Survival Motor Neuron 1 (SMN1) gene, resulting in deficiency of functional SMN protein. The pathologic aspect of SMA is the progressive loss of Motor Neurons (MNs) in the ventral horn of the spinal cord, which causes the loss of muscle function, paralysis and premature death. A first drug for SMA, Nusinersen an antisense oligonucleotide (ASO) has been recently been approved. In fact, one of the most promising strategies in order to increase the SMN protein levels is the use of ASOs to redirect the splicing of the paralogous gene SMN2 to increase the production of the functional SMN protein. ASOs with different chemical structure are currently used in clinical trials, included the ASO with Morpholino (MO) chemistry studied in this work. MO oligomers are particularly suitable for in vivo applications thanks to their optimal safety and efficacy profile. One of the issues related to the administration of ASOs is that they are not able to cross the blood-brain barrier and they must be administered by relatively invasive intrathecal injection to reach the central nervous system (CNS). Therefore, we aimed to improve the efficacy of MO and to develop a non-invasive systemic delivery in an in vivo SMA murine model. First, to increase the efficacy of MO oligomers, we tested, both in vitro and in vivo, four new MO sequences targeting the region involved in the alternative splicing of SMN2 mRNA, called intronic splicing silencer N1 (ISS-N1). Simultaneously, to improve the delivery of MO to affected tissues, we conjugated it to cell-penetrating peptides (CPPs), an approach already used to enhance the cellular and tissue uptake and the pharmacological profile of ASOs, but never explored in SMA. We tested three different CPPs: Tat, R6, and (RXRRBR)2XB, which were linked to our already validated MO sequence [HSMN2Ex7D(-10,-34)]. We IV administered the conjugates in pre-symptomatic and symptomatic SMA mice to determine their therapeutic efficacy versus unconjugated-MO. We showed that optimization of the target sequence can enhance the splice- correction action of MO oligomers and that the effect of the co-administration of different MO sequences is superior to the delivery of a single sequence. Furthermore, we showed that CPP-MO compounds can up-regulate the SMN protein into the CNS of SMA7 transgenic murine models as well as their average lifespan and motor performance after local and systemic injection. Our data proved that the CPP-MO conjugates can be a suitable strategy to increase the cellular uptake after an intrathecal administration and to deliver MO oligomers to the CNS after a systemic injection. The presented results provided the basis for the selection of the most efficient CPPs and will set the stage for future clinical trials. V Index 1. INTRODUCTION ................................................................................. 1 1.1 SPINAL MUSCULAR ATROPHIES ............................................... 1 1.1.1 HISTORY OF THE DESEASE ................................................ 2 1.1.2 CLINICAL CLASSIFICATION ................................................ 3 1.2 GENETIC AND MOLECULAR FINDINGS ..................................... 5 1.2.1 SMN GENE ............................................................................ 6 1.2.2 SMN PROTEIN ..................................................................... 11 1.2.3 ANIMAL MODELS OF SMA ................................................. 12 1.3 DIAGNOSIS ................................................................................. 14 1.3.1 GENETIC COUNSELING AND PRENATAL DIAGNOSIS ... 14 1.3.2 MOLECULAR DIAGNOSIS .................................................. 15 1.4 THERAPEUTIC STRATEGIES FOR SMA ................................... 16 1.4.1 GENE THERAPY ................................................................. 17 1.4.2STEM CELLS THERAPY ...................................................... 19 1.4.3 PHARMACOLOGICAL TREATMENT .................................. 19 1.4.4 SMALL MOLECULES .......................................................... 20 1.4.5 ANTISENSE OLIGONUCLEOTIDES ................................... 21 1.5 DRUG DELIVERY TO THE CENTRAL NERVOUS SYSTEM ...... 27 1.5.1 CROSSING THE BLOOD-BRAIN BARRIER ....................... 27 1.5.2 CELL-PENETRATING PEPTIDES ....................................... 28 1.5.3 SMALL INTERFERING RNA (siRNA) DELIVERY ............... 31 1.5.4 DNA DELIVERY ................................................................... 33 1.5.5 ANTISENSE OLIGONUCLEOTIDES DELIVERY ................. 34 1.5.6 PROTEIN DELIVERY ........................................................... 35 1.6 CLINICAL TRIALS ...................................................................... 36 VI 2. AIM OF THE STUDY ......................................................................... 38 3. MATERIALS AND METHODS .......................................................... 41 3.1 PHOSPHORODIAMIDATE MORPHOLINO OLIGONUCLEOTIDES ........................................................................................................... 41 3.2 CONTROL CELL LINE ................................................................ 42 3.3 REPROGRAMMING OF
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