PRODUZIONE DI CELLULE STAMINALI MESENCHIMALI PER APPLICAZIONI DI TERAPIA AVANZATA

SOMMARIO I prodotti di ingegneria tissutale sono medicinali che contengono o consistono in cellule o tessuti sottoposti ad una rilevante manipolazione cos\uec da ottenere caratteristiche biologiche, funzioni fisiologiche e propriet\ue0 strutturali pertinenti alla finalit\ue0 di rigenerazione, riparazione o sostituzione. Nel caso di cellule e tessuti manipolati in vitro, l'obiettivo da raggiungere nel controllo dei processi di produzione e della qualit\ue0 del prodotto finale \ue8 garantire la sicurezza e l'efficacia dei prodotti da immettere nell'uso clinico. Ne consegue la necessit\ue0 di operare nel rispetto di norme proprie dei processi produttivi dei farmaci sia dal punto di vista della qualit\ue0 che della sicurezza del prodotto. Lo scopo del lavoro svolto durante il dottorato \ue8 stato lo studio e messa a punto di sistemi di produzione per la generazione di un prodotto innovativo per terapie avanzate. Si tratta di un prodotto costituito da cellule staminali mesenchimali, ottime candidate per applicazioni cliniche in medicina rigenerativa. Per la produzione di un prodotto di terapia avanzata, l\u2019intero processo, a partire dal campione iniziale fino al prodotto finito, deve essere svolto in un\u2019officina farmaceutica autorizzata che operi nel rispetto delle Good Manufacturing Practices (GMP). Si tratta di linee guida il cui scopo \ue8 assicurare che un farmaco sia prodotto, analizzato e rilasciato in un regime di Qualit\ue0 controllata e certificata in modo da minimizzare il pericolo che vi siano rischi non previsti per il paziente. Nella fase preliminare del processo \ue8 stata valutata la fattibilit\ue0 del metodo e sono stati definiti i protocolli da impiegare. La fase di fattibilit\ue0 ha permesso di mettere a punto la procedura di isolamento, espansione, differenziamento delle cellule staminali. E\u2019 stato possibile valutare la stabilit\ue0 genomica e le caratteristiche immunofenotipiche delle cellule a vari passaggi cellulari. Tutti i dati ottenuti durante lo studio di fattibilit\ue0 sono stati fondamentali per definire i test di controllo di qualit\ue0, le specifiche di prodotto e i criteri di accettabilit\ue0 richiesti per la successiva convalida del processo. I risultati presentati durante lo studio di fattibilit\ue0 evidenziano come sia possibile trasferire protocolli di ricerca in processi potenzialmente applicabili in sperimentazione clinica. Alla fine del processo di convalida che prevede la produzione di tre lotti di cellule, le specifiche previste per i controlli in ingresso, durante il processo di produzione e sul prodotto finito devono risultare conformi a tutte le richieste. I passi futuri sono la validazione del processo asettico, mediante l\u2019esecuzione di tre mediafill e la valutazione del rischio relativa alla produzione di lotti destinati alla clinica, in modo da completare la serie di studi necessari per la presentazione di una domanda di autorizzazione allo studio clinico.ABSTRACT Tissue Engineered products may carry cells or tissues either of human or animal origin. The cells and tissues shall be subjected to substantial manipulation in order to obtain biological characteristics, physiological functions or structural properties relevant for the intended regeneration, repair or replacement. For cells and tissues manipulated in vitro, the objective to be achieved in terms of control of production processes and quality of the final product is to ensure the safety and effectiveness of the products that would be placed in clinical use. Hence the need to act in accordance with the rules which define production processes used for drugs, in order to guarantee the quality and safery of the product.. The purpose of the work done during the PhD was the study and the development of production protocol for the generation of an innovative product for advanced therapies. It is a cellular product made of mesenchymal stem cells, good candidates for clinical applications in regenerative medicine. For this production, all stages, starting from the initial sample and up to the final product, must be carried out in an authorized pharmaceutical facility which operates in compliance with Good Manufacturing Practices (GMP). The purpose of these guidelines is to ensure that drugs are produced, analysed and released in a regime of controlled and certified quality minimizing the danger of unexpected risks for the patient. In the preliminary phase of the process the feasibility of the method was evaluated and the protocols to be used were defined. The feasibility phase allowed the development of procedures for the isolation, expansion and differentiation of stem cells. It was possible to evaluate the genomic stability and immunophenotypic features of the cells at different steps. All data obtained during the feasibility study have been fundamental to define the tests of quality control, product specifications and criteria of acceptability required for the subsequent validation of the process. The results presented in the feasibility study show that it is possible to transfer research protocols to a GMP framework which is potentially applicable in clinical trials. At the end of the validation process, which involves the production of three batches of cells, the specifications required for the incoming controls, during the production\u2019s process and on the final product must comply with all the requirements. The future steps will be the validation of the aseptic process, through the execution of three mediafill and the risk assessment related to the production of batches intended for clinical use, in order to complete the series of documents required for the submission of an application for a clinical study

Probing The Function Of Long Noncoding RNAs In The Nucleus

The nucleus is a highly organized and dynamic environment where regulation and coordination of processes such as gene expression and DNA replication are paramount. In recent years, noncoding RNAs have emerged as key participants in the regulation of nuclear processes. There are a multitude of functional roles for long noncoding RNA (lncRNA), mediated through their ability to act as molecular scaffolds bridging interactions with proteins, chromatin, and other RNA molecules within the nuclear environment. In this review, we discuss the diversity of techniques that have been developed to probe the function of nuclear lncRNAs, along with the ways in which those techniques have revealed insights into their mechanisms of action. Foundational observations into lncRNA function have been gleaned from molecular cytology-based, single-cell approaches to illuminate both the localization and abundance of lncRNAs in addition to their potential binding partners. Biochemical, extraction-based approaches have revealed the molecular contacts between lncRNAs and other molecules within the nuclear environment and how those interactions may contribute to nuclear organization and regulation. Using examples of well-studied nuclear lncRNAs, we demonstrate that the emerging functions of individual lncRNAs have been most clearly deduced from combined cytology and biochemical approaches tailored to study specific lncRNAs. As more functional nuclear lncRNAs continue to emerge, the development of additional technologies to study their interactions and mechanisms of action promise to continually expand our understanding of nuclear organization, chromosome architecture, genome regulation, and disease states

Cost-effectiveness of catheter ablation versus medical therapy for the treatment of atrial fibrillation in the United Kingdom.

INTRODUCTION: Research evidence has shown that catheter ablation is a safe and superior treatment for atrial fibrillation (AF) compared to medical therapy, but real-world practice has been slow to adopt an early interventional approach. This study aims to determine the cost effectiveness of catheter ablation compared to medical therapy from the perspective of the United Kingdom. METHODS: A patient-level Markov health-state transition model was used to conduct a cost-utility analysis. The population included patients previously treated for AF with medical therapy, including those with heart failure (HF), simulated over a lifetime horizon. Data sources included published literature on utilization and cardiovascular event rates in real world patients, a systematic literature review and meta-analysis of randomized controlled trials for AF recurrence, and publicly available government data/reports on costs. RESULTS: Catheter ablation resulted in a favorable incremental cost-effectiveness ratio (ICER) of £8614 per additional quality adjusted life years (QALY) gained when compared to medical therapy. More patients in the medical therapy group failed rhythm control at any point compared to catheter ablation (72% vs. 24%) and at a faster rate (median time to treatment failure: 3.8 vs. 10 years). Additionally, catheter ablation was estimated to be more cost-effective in patients with AF and HF (ICER = £6438) and remained cost-effective over all tested time horizons (10, 15, and 20 years), with the ICER ranging from £9047-£15 737 per QALY gained. CONCLUSION: Catheter ablation is a cost-effective treatment for atrial fibrillation, compared to medical therapy, from the perspective of the UK National Health Service

MIR-206 regulates connexin43 expression during skeletal muscle development

Skeletal myoblast fusion in vitro requires the expression of connexin43 (Cx43) gap junction channels. However, gap junctions are rapidly downregulated after the initiation of myoblast fusion in vitro and in vivo. In this study we show that this downregulation is accomplished by two related microRNAs, miR-206 and miR-1, that inhibit the expression of Cx43 protein during myoblast differentiation without altering Cx43 mRNA levels. Cx43 mRNA contains two binding sites for miR-206/miR-1 in its 3′-untranslated region, both of which are required for efficient downregulation. While it has been demonstrated before that miR-1 is involved in myogenesis, in this work we show that miR-206 is also upregulated during perinatal skeletal muscle development in mice in vivo and that both miR-1 and miR-206 downregulate Cx43 expression during myoblast fusion in vitro. Proper development of singly innervated muscle fibers requires muscle contraction and NMJ terminal selection and it is hypothesized that prolonged electrical coupling via gap junctions may be detrimental to this process. This work details the mechanism by which initial downregulation of Cx43 occurs during myogenesis and highlights the tight control mechanisms that are utilized for the regulation of gap junctions during differentiation and development

Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements.

Background: Mutations in gene regulatory networks often lead to genetic divergence without impacting gene expression or developmental patterning. The rules governing this process of developmental systems drift, including the variable impact of selective constraints on different nodes in a gene regulatory network, remain poorly delineated. Results: Here we examine developmental systems drift within the cardiopharyngeal gene regulatory networks of two tunicate species, Corella inflata and Ciona robusta. Cross-species analysis of regulatory elements suggests that trans-regulatory architecture is largely conserved between these highly divergent species. In contrast, cis-regulatory elements within this network exhibit distinct levels of conservation. In particular, while most of the regulatory elements we analyzed showed extensive rearrangements of functional binding sites, the enhancer for the cardiopharyngeal transcription factor FoxF is remarkably well-conserved. Even minor alterations in spacing between binding sites lead to loss of FoxF enhancer function, suggesting that bound trans-factors form position-dependent complexes. Conclusions: Our findings reveal heterogeneous levels of divergence across cardiopharyngeal cis-regulatory elements. These distinct levels of divergence presumably reflect constraints that are not clearly associated with gene function or position within the regulatory network. Thus, levels of cis-regulatory divergence or drift appear to be governed by distinct structural constraints that will be difficult to predict based on network architectur

Variable Levels Of Drift In Tunicate Cardiopharyngeal Gene Regulatory Elements

Background: Mutations in gene regulatory networks often lead to genetic divergence without impacting gene expression or developmental patterning. The rules governing this process of developmental systems drift, including the variable impact of selective constraints on different nodes in a gene regulatory network, remain poorly delineated. Results: Here we examine developmental systems drift within the cardiopharyngeal gene regulatory networks of two tunicate species, Corella inflata and Ciona robusta. Cross-species analysis of regulatory elements suggests that trans-regulatory architecture is largely conserved between these highly divergent species. In contrast, cis-regulatory elements within this network exhibit distinct levels of conservation. In particular, while most of the regulatory elements we analyzed showed extensive rearrangements of functional binding sites, the enhancer for the cardiopharyngeal transcription factor FoxF is remarkably well-conserved. Even minor alterations in spacing between binding sites lead to loss of FoxF enhancer function, suggesting that bound trans-factors form position-dependent complexes. Conclusions: Our findings reveal heterogeneous levels of divergence across cardiopharyngeal cis-regulatory elements. These distinct levels of divergence presumably reflect constraints that are not clearly associated with gene function or position within the regulatory network. Thus, levels of cis-regulatory divergence or drift appear to be governed by distinct structural constraints that will be difficult to predict based on network architecture

Prevalence of transmitted nucleoside analogue-resistant HIV-1 strains and pre-existing mutations in pol reverse transcriptase and protease region : outcome after treatment in recently infected individuals

We retrospectively studied 38 Italian recently HIV-1-infected subjects who seroconverted from 1994 to 1997 to investigate: (i) the prevalence of nucleoside reverse transcriptase inhibitors (NRTI)-related mutations at primary infection; (ii) the proportion of naturally occurring mutations in reverse transcriptase (RT) and protease regions of patients naive for non-nucleoside RT inhibitors (NNRTIs) and protease inhibitors (PIs); (iii) the drug-susceptibility to NRTIs and PIs in subjects with NRTI- and/or PI-related mutations; and (iv) the outcome of seroconverters treated with various NRTIs or NRTI/PI regimens. Baseline HIV-1 plasma viraemia and absolute CD4 count at baseline could not be used to distinguish patients with NRTI- and/or PI-related pre-existing mutations from those with wild-type virus (P = 0.693 and P = 0.542, respectively). The frequency of zidovudine-related mutations was 21% in the study period. The response to treatment was not significantly different in subjects with or without genotypic zidovudine-related mutations at primary infection (P = 0.744 for HIV-1 RNA and P = 0.102 for CD4 cells). Some natural variation (2.6%) was present within regions 98-108 and 179-190 of RT involved in NNRTI resistance. The high natural polymorphism in the protease region present in our patients was similar to that reported by others. In our study some PI-associated substitutions, thought to be compensatory in protease enzymatic function, could confer intermediate to high PI-resistance. As discrepancies between genotypic and phenotypic results may exist in recent seroconverters, our data suggest that the role of transmitted NRTI- and PI-resistant variants remain to be fully elucidated in vivo

Identification of two distinct subsets of long-term nonprogressors with divergent viral activity by stromal-derived factor 1 chemokine gene polymorphism analysis

Stromal-derived factor (SDF)-1, the natural ligand for CXCR4, is present in a common polymorphic variant defined by a G-->A transition in the 3' untranslated region of the gene. In persons infected with human immunodeficiency virus type 1 (HIV-1), the homozygous genotype (SDF1-3'A/3'A) has been postulated to interfere with the appearance of T-tropic syncytium-inducing strains. The polymorphism of SDF1 was correlated with HIV-1 phenotype, plasma viremia, and unspliced and multiply spliced specific transcripts in 158 virologically characterized HIV-1-infected patients (39 recent seroconverters, 75 typical progressors, and 44 AIDS patients) and in 42 HIV-1-infected long-term nonprogressors (LTNPs). Analysis of SDF1 allele distribution revealed that SDF1-3'A/3'A status is associated with low CD4 cell count (P=.0449) but not with a specific HIV-1 phenotype. In LTNPs, SDF1-+/+ condition defined a subset of persons with lower HIV-1 replication than in heterozygous subjects. The low viral activity in SDF1-+/+ LTNPs suggests that other factors play a major role in vivo in determining the course of HIV-1 infection