Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

Ruolo del gene umano CYYR1: dallo studio funzionale dell'ortologo in Danio rerio alla potenziale implicazione in processi tumorigenici

Il locus CYYR1 identificato e clonato sul cromosoma 21 umano è stato caratterizzato dal punto di vista molecolare come un sistema multitrascritto, esclusivo dei vertebrati che ad oggi è orfano di una funzione specifica. Dati presenti in lettura e rintracciati mostrano una possibile relazione tra il gene CYYR1 e il pathway di Sonic Hedgehog (SHH). In questo progetto di tesi è stato utilizzato il modello animale Danio rerio per indagare il ruolo funzionale dell’ortologo (cyyr1), attraverso esperimenti di gain e loss of function che hanno permesso di dimostrare un suo coinvolgimento nello sviluppo del sistema nervoso centrale, del cuore e del tessuto muscolare. Lo studio dell’ortologo in zebrafish è stato associato all’utilizzo di linee cellulari di rabdomiosarcoma umano. I risultati ottenuti dall’induzione al differenziamento miogenico di queste linee, insieme ai dati ottenuti in Danio rerio, confermano il possibile coinvolgimento del gene CYYR1 nella miogenesi. Lo studio delle relazione tra il pathway di SHH e l’espressione del gene CYYR1 è stato condotto in entrambi i modelli con l’utilizzo di differenti inibitori della via di segnalazione. I risultati ottenuti mostrano che sistemi inibitori agenti direttamente sul recettore SMO riducono l’espressione del gene. Un dato inaspettato in Danio rerio ottenuto durante questi esperimenti di inibizione, ha aperto una nuova linea di ricerca in collaborazione con l’Università di Warwick tesa a verificare la relazione tra il gene cyyr1 e il gene lefty1. Gli esperimenti condotti presso il laboratorio della Prof.ssa Sampath hanno dimostrato la localizzazione del prodotto proteico cyyr1 in Danio rerio e indagato co-localizzazioni con la proteina lefty1. Infine, in collaborazione con Dr. Deflorian e della Prof.ssa Pistocchi, è stato generato un mutante di Danio rerio deleto per il gene cyyr1 con la tecnica CRISPR/Cas9. La caratterizzazione del mutante cyyr1 -/- ha confermato alcuni dei dati ottenuti attraverso esperimenti di loss of function.

Biodegradable systems for the development of functional materials

This PhD work was aimed to design, develop, and characterize gelatin-based scaffolds, for the repair of defects in the muscle-skeletal system. Gelatin is a biopolymer widely used for pharmaceutical and medical applications, thanks to its biodegradability and biocompatibility. It is obtained from collagen via thermal denaturation or chemical-physical degradation. Despite its high potential as biomaterial, gelatin exhibits poor mechanical properties and a low resistance in aqueous environment. Crosslinking treatment and enrichment with reinforcement materials are thus required for biomedical applications. In this work, gelatin based scaffolds were prepared following three different strategies: films were prepared through the solvent casting method, electrospinning technique was applied for the preparation of porous mats, and 3D porous scaffolds were prepared through freeze-drying. The results obtained on films put into evidence the influence of pH, crosslinking and reinforcement with montmorillonite (MMT), on the structure, stability and mechanical properties of gelatin and MMT/gelatin composites. The information acquired on the effect of crosslinking in different conditions was utilized to optimize the preparation procedure of electrospun and freeze-dried scaffolds. A successful method was developed to prepare gelatin nanofibrous scaffolds electrospun from acetic acid/water solution and stabilized with a non-toxic crosslinking agent, genipin, able to preserve their original morphology after exposure to water. Moreover, the co-electrospinning technique was used to prepare nanofibrous scaffolds at variable content of gelatin and polylactic acid. Preliminary in vitro tests indicated that the scaffolds are suitable for cartilage tissue engineering, and that their potential applications can be extended to cartilage-bone interface tissue engineering. Finally, 3D porous gelatin scaffolds, enriched with calcium phosphate, were prepared with the freeze-drying method. The results indicated that the crystallinity of the inorganic phase influences porosity, interconnectivity and mechanical properties. Preliminary in vitro tests show good osteoblast response in terms of proliferation and adhesion on all the scaffolds.

Development of strategies for vascular damage repair in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive and rare disease with so far unclear pathogenesis, limited treatment options and poor prognosis. Unbalance of proliferation and migration in pulmonary arterial smooth muscle cells (PASMCs) is an important hallmark of PAH. In this research Sodium butyrate (BU) has been evaluated in vitro and in vivo models of PAH. This histone deacetylase inhibitor (HDACi) counteracted platelet-derived growth factor (PDGF)-induced ki67 expression in PASMCs, and arrested cell cycle mainly at G0/G1 phases. Furthermore, BU reduced the transcription of PDGFRbeta, and that of Ednra and Ednrb, two major receptors in PAH progression. Wound healing and pulmonary artery ring assays indicated that BU inhibited PDGF-induced PASMC migration. BU strongly inhibited PDGF-induced Akt phosphorylation, an effect reversed by the phosphatase inhibitor calyculinA. In vivo, BU showed efficacy in monocrotaline-induced PAH in rats. Indeed, the HDACi reduced both thickness of distal pulmonary arteries and right ventricular hypertrophy. Besides these studies, Serial Analysis of Gene Expression (SAGE) has be used to obtain complete transcriptional profiles of peripheral blood mononuclear cells (PBMCs) isolated from PAH and Healthy subjects. SAGE allows quantitative analysis of thousands transcripts, relying on the principle that a short oligonucleotide (tag) can uniquely identify mRNA transcripts. Tag frequency reflects transcript abundance. We enrolled patients naïve for a specific PAH therapy (4 IPAH non-responder, 3 IPAH responder, 6 HeritablePAH), and 8 healthy subjects. Comparative analysis revealed that significant differential expression was only restricted to a hundred of down- or up-regulated genes. Interestingly, these genes can be clustered into functional networks, sharing a number of crucial features in cellular homeostasis and signaling. SAGE can provide affordable analysis of genes amenable for molecular dissection of PAH using PBMCs as a sentinel, surrogate tissue. Altogether, these findings may disclose novel perspectives in the use of HDACi in PAH and potential biomarkers.

Electrospun hierarchical artificial muscle for soft robotics applications

Nowadays, one of the most ambitious challenges in soft robotics is the development of actuators capable to achieve performance comparable to skeletal muscles. Scientists have been working for decades, inspired by Nature, to mimic both their complex structure and their perfectly balanced features in terms of linear contraction, force-to-weight ratio, scalability and flexibility. The present Thesis, contextualized within the FET open Horizon 2020 project MAGNIFY, aims to develop a new family of innovative flexible actuators in the field of soft-robotics. For the realization of this actuator, a biomimetic approach has been chosen, drawing inspiration from skeletal muscle. Their hierarchical fibrous structure was mimicked employing the electrospinning technique, while the contraction of sarcomeres was designed employing chains of molecular machines, supramolecular systems capable of performing movements useful to execute specific tasks. The first part deals with the design and production of the basic unit of the artificial muscle, the artificial myofibril, consisting in a novel electrospun core-shell nanofiber, with elastomeric shell and electrically conductive core, coupled with a conductive coating, for the realization of which numerous strategies have been investigated. The second part deals instead with the integration of molecular machines (provided by the project partners) inside these artificial myofibrils, preceded by the study of several model molecules, aimed at simulating the presence of these molecular machines during the initial phases of the project. The last part concerns the realization of an electrospun multiscale hierarchical structure, aimed at reproducing the entire muscle morphology and fibrous organization. These research will be joined together in the near future like the pieces of a puzzle, recreating the artificial actuator most similar to biological muscle ever made, composed of millions of artificial myofibrils, electrically activated in which the nano-scale movement of molecular machines will be incrementally amplified to the macro-scale contraction of the artificial muscle.