Retroviral Integrations in Gene Therapy Trials

γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis

Identification and validation of molecular biomarkers in Ewing sarcoma

ll sarcoma di Ewing, secondo più diffuso tumore primitivo dell’osso, è un tumore di origine mesenchimale che colpisce prevalentemente adolescenti e giovani adulti. L’attuale terapia multimodale ha raggiunto un plateau di sopravvivenza a 5 anni (5y OVS) pari al 70% per pazienti con malattia localizzata, 30% e 25% per pazienti metastatici alla diagnosi o recidivanti. Poichè inoltre i pazienti soffrono di tossicità acuta o a lungo termine legata alla chemioterapia, è fondamentale lo sviluppo di biomarcatori che ne permettano la stratificazione in base al rischio alla diagnosi. Questo studio conferma LGALS3BP e miR-34a come predittori indipendenti dell’esito clinico in 124 pazienti affetti da sarcoma di Ewing localizzato trattati presso un singolo Istituto. Pazienti alto-esprimenti hanno un rischio ridotto di sviluppare recidive locali o metastasi (HR 0.402, 95% CI 0.190-0.850 per LGALS3BP; HR 0.485, 95% CI 0.236 -0.996 per miR-34a), o di decesso legato al tumore (HR 0.397, 95% CI 0.159-0.993 per LGALS3BP; HR 0.353, 95% CI 0.132-0.943 per miR-34a). Tramite lo sviluppo e la caratterizzazione in vitro ed in vivo di un modello di over-espressione stabile di miR-34a, abbiamo confermato il ruolo oncosoppressore di questo miRNA nel sarcoma di Ewing; uno dei meccanismi sembra coinvolgere l’inibizione dell’isoforma 1B della ciclina D1, target diretto di EWS-FLI1, fattore trascrizionale aberrante caratteristico di questo tumore. È stata inoltre provata la fattibilità della quantificazione non invasiva di miR-34a circolante. Tramite whole transcriptome sequencing su biopsie pre-trattamento da pazienti selezionati in base alla differenziale risposta alla chemioterapia, sono state identificate 4 mutazioni a carico di TP53 (p.R213X, p.R248W, p.R273C ep.K386M mutazione de novo), che definiscono una classe di pazienti con scarsa risposta al trattamento, ed un pannello di lincRNAs con espressione differenziale nei due gruppi. Nell’insieme questi risultati concorrono alla definizione di uno spettro di caratteristiche molecolari che riteniamo promettenti candidati per la validazione prospettica.Ewing sarcoma, the second most common primary bone malignancy, is a highly aggressive mesenchymal tumor arising mainly in adolescents and young adults. Current multimodal treatment has reached a plateau of 70% 5-year overall survival (5y OVS) for patients with localized disease, and 30% and 25% for patients with metastatic or recurrent disease. Moreover, patients often suffer from acute and long term toxicity related to chemotherapy. It is thus paramount the development of molecular biomarkers allowing stratification of patients based on risk at diagnosis. The present study validates LGALS3BP and miR-34a as independent predictors of clinical outcome in 124 Ewing sarcoma patients treated in a single Institution. Patients with high expression levels of these molecules have lower risk of developing local recurrences or metastasis (HR 0.402, 95% CI 0.190-0.850 for LGALS3BP;HR 0.485, 95% CI 0.236 -0.996 for miR-34a), and a lower risk of disease-related death (HR 0.397, 95% CI 0.159- 0.993 for LGALS3BP; HR 0.353, 95% CI 0.132-0.943 for miR-34a). By developing and characterizing in vitro and in vivo a stable miR34a expression model, we confirmed its oncosuppressor role in Ewing sarcoma; one of the underlying mechanisms involve the inhibition of the 1B isoform of cyclin D1, a direct target of EWS-FLI1, the aberrant transcription factor characteristic of the disease. We also show the feasibility of non-invasive circulating miR-34a quantification. Through a whole transcriptome sequencing approach applied to pre-treatment biopsies from patients with differential chemotherapy response, we identified 4 mutations in the TP53 gene (p.R213X, p.R248W, p.R273C and p.K386M de novo mutation) defining a patient group with poor response to treatment, and a panel of lincRNA with differential expression in the two groups. Overall these results contribute to the definition of a spectrum of molecular features of Ewing sarcoma impacting clinical outcome, which are promising candidates for prospective validation

Leucocyte morphology and chromosome morphology

The banding techniques currently employed in human cytogenetics for the identification of the individual chromosomes have been used to stain PHA lymphocytes and circulating leucocytes. The capacity of these techniques to localize singular chromosomes or chromosomal regions has been investigated. It has been observed that among the four major categories of bands (Q, G, E and R) only the quinacrine staining is informative in interphase nuclei, because of its peculiarity to stain the long arm of the Y chromosome and few other heterochromatic regions. Interphase nuclei treated according to the C bands show the presence of several heterochromatic masses, corresponding to the centromeric areas of individual chromosomes, but as such they cannot be recognized accurately. More specific and selective techniques, like G 11 and G Y protocols, appear to be suitable to localize the centromeric regions of chromosome no. 9 and the long arm of Y chromosome. Variation of the incubation time in the alkali saline solutions and of pH values have proven to be appropriate for the demonstration of other heterochromatic regions in interphase nuclei and in circulating leucocytes. The 'nuclear' approach to the study of specific heterochromatic regions of human chromosomes may be of practical interest into the investigation of several biological problems and into the detection of individuals carrying chromosome variants

IS-Seq: a bioinformatics pipeline for integration sites analysis with comprehensive abundance quantification methods

Abstract Background Integration site (IS) analysis is a fundamental analytical platform for evaluating the safety and efficacy of viral vector based preclinical and clinical Gene Therapy (GT). A handful of groups have developed standardized bioinformatics pipelines to process IS sequencing data, to generate reports, and/or to perform comparative studies across different GT trials. Keeping up with the technological advances in the field of IS analysis, different computational pipelines have been published over the past decade. These pipelines focus on identifying IS from single-read sequencing or paired-end sequencing data either using read-based or using sonication fragment-based methods, but there is a lack of a bioinformatics tool that automatically includes unique molecular identifiers (UMI) for IS abundance estimations and allows comparing multiple quantification methods in one integrated pipeline. Results Here we present IS-Seq a bioinformatics pipeline that can process data from paired-end sequencing of both old restriction sites-based IS collection methods and new sonication-based IS retrieval systems while allowing the selection of different abundance estimation methods, including read-based, Fragment-based and UMI-based systems. Conclusions We validated the performance of IS-Seq by testing it against the most popular  analytical workflow available in the literature (INSPIIRED) and using different scenarios. Lastly, by performing extensive simulation studies and a comprehensive wet-lab assessment of our IS-Seq pipeline we could show that in clinically relevant scenarios, UMI quantification provides better accuracy than the currently most widely used sonication fragment counts as a method for IS abundance estimation

Monoclonal antibodies against HLA class I antigens inhibit human lymphocyte proliferation but do not affect mitogen-dependent second messenger generation

The mechanism through which anti HLA class I monoclonal antibodies inhibit human lymphocyte proliferation induced by the policlonal mitogen phytohemoagglutinin was investigated. Anti HLA class I monoclonal antibodies inhibited mitogen stimulated DNA synthesis even when added several hours after phytohemoagglutinin. The extent of inhibition depended on the duration of the exposure of lymphocytes to the monoclonal antibodies. Anti HLA class I monoclonal Antibodies neither affected the rise in cytosolic fee Ca2+ concentration nor the production of inositol phosphates induced by phytohemoagglutinin. These results demonstrate that anti HLA class I monoclonal antibodies inhibit lymphocyte proliferation at a step downhill of second messenger generation