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Responsabile Antonia Follenzi
Settore di ricerca Istologia
Personale docente Maria Prat  Lia Rimondini 
Personale non-docente Simone Merlin  Stefania Cannizzo   Gabriella Ranaldo  Diego Zanolini  Maria Talmon  Grosso Chantal   Alessio Stevano  Stefano Porta
Obiettivi della ricerca 1) Use of patient-specific induced pluripotent stem cells to improve diagnosis and treatment of hemophilia A
Risultati ottenuti 1) Development of a novel strategy for HA treatment, generating human B-domain-deleted FVIII (hBDD-FVIII)-corrected patient-specific iPSCs and differentiating them into functional endothelial cells (ECs), secreting FVIII in hemophilic mice after transplantation.Reprogrammed MNC gave rise to iPSCs in about 45 days. iPSCs displayed embryonic stem cells (ESC)-like morphology: colonies were compact, uniform and with defined borders when grown on feeders. iPSCs were positive for AP staining and expressed specific stem cell markers at RNA and protein level, showing activation of the endogenous reprogramming factors. Moreover, iPSCs differentiated in osteogenic, condrogenic and adipose tissues. EB expressed markers of the three germ layers: alpha-fetoprotein (endoderm), brachiury (mesoderm) and nestin (ectoderm). Nevertheless, iPSCs showed demethylation at CpG islands at the core of Oct4 promoter, epigenetic marker of complete reprogramming to a pluripotent state. Additionally, telomeres length of original and reprogrammed cells did not show changes indicating telomerase reactivation. Furthermore, RT-PCR on HA-iPSCs showed the expression of hBDD-FVIII, confirming genetic correction of HA-MNC by LV transduction.
Importantly, iPSCs differentiated into ECs, acquiring a typical endothelial-like morphology with an increased expression of ECs markers, such as CD31, KDR, vWF and FVIII. Then, iPSC-derived ECs were transduced with LV-expressing GFP under the control of Flk-1 and Tie2. Flk-1-GFP+ cells were transplanted in NOD-SCID HA mice. GFP+ cells were detected in liver sections up to 1 week post transplantation. Overall, these data will be instrumental to assess the engraftment, the proliferation and the levels of FVIII expression from differentiated, gene corrected and reprogramming factor free iPSCs to confirm the suitability of this approach for HA gene-cell-therapy.
 
Obiettivi della ricerca 2) Strategies to overcome immunological responses to gene therapy
for Hemophilia A
Risultati ottenuti 2) Liver is known to induce tolerance rather than immunity towards antigens that are locally presented to T cells. Although prior efforts in liver-directed gene therapy largely focused on hepatocytes, recently we became interested in liver sinusoidal endothelial cells (LSEC) after studies showing that LSEC can function as resident antigen presenting cells and induce tolerance toward antigens delivered to the liver.We engineered LSEC by LV expressing GFP or hFVIII. In the latest years the immunological role of LSEC has been elucidated and it seems they are able to trigger tolerogenic responses rather than immune responses to several antigens. Moreover, to overcome potential immunological problems, we modified lentiviral constructs in order to limit transgene expression exclusively in endothelial cells by transcriptional and post-transcriptional regulation. We initially included an endothelial specific promoter, the vascular endothelial cadherin promoter and later we included in the construct a layer of post transcriptional control as well, mediated by endogenous microRNA (miRNA) to detarget transgene expression in target cells. Adding a miRT122 and 142-3p sequences we were able to restrict the expression in EC. In our study, post-transcriptional modification and transcriptional targeting with endothelial specific–promoter were able to overcome off-target expression of GFP and FVIII in liver cells. Incorporation of miRNA regulation into a LV vector was able to provide an important layer of control on transgene expression. We would like to speculate that restricting FVIII expression in LSEC and de-targeting expression in conventional APC would prevent immune responses to the secreted factor. Antigen presentation by nonprofessional APC, such as LSEC, may provide a pathway for achieving antigen-specific tolerance. Further studies will be necessary to elucidate this issue. 
Obiettivi della ricerca 3) Trapianto di cellule di Kupffer per la correzione del fenotipo emorragico dell`emofilia A
Risultati ottenuti 3) Le cellule di Kupffer (KC) rappresentano la maggior parte dei macrofagi dell’organismo. Sono pochi i dati sulla biologia delle KC, in particolar modo quelli che riguardano la loro origine, il loro turnover e il loro rimpiazzo e/o manipolazione a scopo terapeutico. Il nostro studio è diretto a capire se il il trapianto di KC o di macrofagi derivati da midollo osseo (BMDM) è in grado di rimpiazzare il compartimento delle KC native. A questo scopo sono state isolate KC murine e umane, mentre cellule del midollo osseo sono state differenziate in macrofagi in vitro. Le cellule ottenute, caratterizzate in vitro, sono state quindi trapiantate. Tramite radiomarcatura e l’utilizzo di geni reporter abbiamo seguito la biodistribuzione e l’attecchimento delle cellule trapiantate. KC e BMDM hanno dimostrato di esprimere marcatori macrofagici e di possedere attività fagocitica. Le KC trapiantate hanno attecchito e sono sopravvissute fino a 3 mesi dopo il trapianto nel fegato dei topi riceventi pre-trattati con gadolinio. Trattamenti capaci di indurre proliferazione a livello epatico, quali epatectomia parziale o iniezione di tetracloruro di carbonio, non hanno avuto effetti sulla proliferazione delle cellule trapiantate, dimostrando una loro lunga sopravvivenza piuttosto che un loro autorinnovo. Attraverso RT-PCR e immunofluorescenza abbiamo dimostrato l’espressione di FVIII da parte delle KC umane e murine. Inoltre, esperimenti condotti a breve termine hanno dimostrato che topi emofilici sono sopravvissuti al tail clip challenge dopo iniezione di KC sane. Le KC trapiantate mantengono attività fagocitica e capacità di produrre citochine. Al contrario, i BMDM non hanno attecchito e sono stati eliminati entro 24 ore dopo il trapianto. Questi studi hanno dimostrato che le KC hanno una lunga vita e che il trapianto di KC sane in topi emofilici è in grado di correggerne il fenotipo emorragico in esperimenti a breve termine. Ulteriori approcci di terapia cellulare e genica per rimpiazzare il compartimento di KC possono aiutare a comprenderne la biologia e il loro potenziale terapeutico.  
Collaborazioni in atto With Sanjeev Gupta, MD; Laura Santambrogio,MD, PhD; Robert Singer, PhD at Albert Einstein College of Medicine of Yeshiva University, Bronx, NY.
Nancy Carrasco, MD (Yale, New Haven, CT), Yelena Ginzburg, MD (New York Blood Center, New York, NY), Ombretta Salvucci, PhD (NCI/NIH, Bethesda, MD),  
Comunicazioni a congressi 1) Engineered cell sheets using thermo-reversible hydrogel. Cochis, A.; Carletta, A.; Altomare, L.; Fare, S; Merlin, S ; Pietronave, S ; Follenzi, A ; Prat, M; Rimondini, L. JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE Volume: 6 Special Issue: SI Supplement: 1 Pages: 203-204 Published: SEP 2012
2) FVIII Expression and Secretion in Circulating Blood Cell Types Capable of Correcting Bleeding in Hemophilia A Mice. Merlin,S; Zanolini,D; Ranaldo,G; Feola,M ; Cannizzo,ES ; Valente,G; Prat,M ; Gupta,S; Follenzi, A MOLECULAR THERAPY Vol: 20 Suppl: 1 S198-S199 ASGCT Meeting, Abstract: 514 Published: MAY 2012
3)A Novel iPSC-Based Strategy To Correct the Bleeding Phenotype in Hemophilia A
Ranaldo, G ; Richaud-Patin, Y ; Lombardo, A ; Grosso, C ; Talmon, M ; Raya, A ; Naldini, L; Schinco,P ; Follenzi,A. MOLECULAR THERAPY Vol: 20, Suppl 1, S251-S251 ASGCT Meeting Abstract: 651, Published: MAY 2012
4) ENGRAFTMENT AND PROLIFERATION OF TRANSPLANTED HUMAN HEPATIC STELLATE CELLS IN THE LIVER OF IMMUNODEFICIENT MICE Benten, D; Kluwe, J; Wirth, J ; Kumaran, V; Joseph, B ; Volz, T; Lutgehethmann, M; Bhargava, KK; Cheng, K; Follenzi, A; Gupta, S. JOURNAL OF HEPATOLOGY Vol: 56 Suppl: 2 S146-S147 EASL Meeting Abstract: 361 PublISHED: APR 2012
 
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