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605 The Infusion of Suicide Gene Modified Donor T Cells after Hematopoietic Stem Cell Transplantation Prompts Thymic Renewal in Adult Patients by an IL 7 Dependent Mechanism Molecular Therapy Volume 1[.]
CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION 603 ProSavin® a Gene Therapy Approach for the Treatment of Parkinson Disease: Phase I Clinical Trial Update Kyriacos Mitrophanous,1 Bechir Jarraya,2,3 Scott Ralph,1 Helene Lepetit,2 James Miskin,1 Jean-Marc Gurruchaga,2 Gilles Fenelon,2 Sabrina Boulet,1 Caroline Jan,2 Gilles Bonvento,2 Pierre Brugiere,2 Susan Kingsman,1 Stuart Naylor,1 Philippe Hantraye,2,3 Philippe Remy,2 Stephane Palfi.2,3 Oxford BioMedica (UK) Ltd, Oxford, United Kingdom; 2Henri Mondor Hospital, Paris, France; 3CEA-CNRS MirCen, Fontenay aux Roses, France Parkinson’s disease (PD) is a debilitating neurodegenerative condition that results from the destruction of dopamine-producing (dopaminergic) neurons in the substantia nigra The loss of dopamine, a key neurotransmitter involved in coordinating motor control, results in movement abnormalities such as bradykinesia (slowness of movement), rigidity and postural instability L-Dopa and dopamine agonists provide the primary standard of care for PD and are highly efficacious in the early stages of disease However their long term use is associated with severe motor side effects that seriously impact on the quality of life These side effects are believed to be caused by the fluctuating nature of dopaminergic stimulation that arises from oral drug administration We have developed a lentiviral vector (ProSavin®) derived from the equine infectious anaemia virus expressing the three key dopamine biosynthetic enzymes (tyrosine hydroxylase, aromatic L-amino acid decarboxylase and GTP cyclohydrolase-1), with the aim of providing a continous source of dopamine in the striatum ProSavin was previously demonstrated to mediate dopamine production and behavioural correction in rat and non-human primate models of PD A phase I, open label clinical study has been initiated in which nine PD patients have received ProSavin in three cohorts in the dose escalation part of the trial All the patients have completed at least months follow up and the first cohort are now approaching their thrid year post treatment ProSavin has been demonstrated to be safe and well tolerated at both doses There were no “OFF” state dyskinesias, no immune responses to ProSavin and no serious adverse events Furthermore, encouraging signs of efficacy have been observed on a number of endpoints including UDRS Part IIII, patient diary and quality of life measures An update on the trial and future plans for ProSavin® will be presented 604 Gene Therapy with Lentiviral Vector Transduced CD34+ Cells for the Treatment of Wiskott-Aldrich Syndrome Samantha Scaramuzza,1 Francesca Ferrua,2 Maria Carmina Castiello,1 Luca Biasco,1 Marita Bosticardo,1 Costanza Evangelio,2 Maria Pia Cicalese,2 Miriam Casiraghi,2 Anna Ripamonti,1 Stefania Giannelli,1 Monica Salomoni,3 Andrea Finocchi,4 Alessandra Biffi,1,2 Fabio Ciceri,5 Anna Villa,1,6 Maria Grazia Roncarolo,1,7 Luigi Naldini,1,7 Alessandro Aiuti.1,4 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 2Pediatric Immunohematology and BMT Unit, Scientific Institute HS Raffaele, Milan, Italy; 3MolMed Spa, Milan, Italy; 4Univ of Rome “Tor Vergata”, Rome, Italy; 5Div of Hematology, Scientific Institute HS Raffaele, Milan, Italy; 6IRGBCNR, Cagliari, Italy; 7Università Vita Salute, San Raffaele, Milan, Italy Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, autoimmunity and lymphomas Gene therapy with ex vivo transduced autologous hematopoietic stem cells (HSC) could represent a valid therapeutic alternative to allogenic HSC transplant We previously demonstrated that a lentiviral vector (LVV) encoding for human WAS under the control of an homologous 1.6 kb promoter efficiently corrected human and Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy mouse cells We set up and validated a transduction protocol for CD34+ cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade purified LVV We obtained robust transduction of patients’ progenitor cells, expressing WASp after differentiation to megakaryocytes, without evidence of toxicity Studies in immunodeficient mice showed that human transduced CD34+ cells were able to normally engraft and differentiate towards lymphoid and myeloid cells Analyses of vector integrations showed polyclonal integration patterns in vitro and in engrafted cells in vivo; vector mobilization to host cells and germline transmission of the LVV were excluded by several molecular assays On the basis of these results and preclinical studies in the WAS-KO mouse, a phase I/II gene therapy protocol was opened in April 2010 Patients will receive preconditioning with anti-CD20 mAb and reduced intensity busulfan and fludarabin; ATG will be included in case of autoimmune manifestations The first enrolled patient was a 5.7 year old boy lacking an HLA-compatible donor He was treated with autologous BM and MPB derived transduced CD34+ cells, showing a vector copy number (VCN) of 1.4 and 1.9 respectively, with high gene transfer efficiency (88-92% in clonogenic progenitors) The patient did not experience toxicity, recovered well from transient neutropenia and is currently well, independent from platelet transfusions, months after gene therapy Engraftment analyses revealed the presence of transduced cells at substantial levels in peripheral blood granulocytes and monocytes (VCN: 0.3-0.4) and at higher levels in T, B and NK lymphocytes (VCN range 0.5-1.0) as expected from the selective advantage Engraftment was present in multiple lineages of the bone marrow (VCN range 0.2-0.9), including CD34+ cells and clonogenic progenitors (25% transd.) WASp expression was detected in peripheral blood monocytes, platelets and all lymphocyte lineages Long-term studies will provide key information on the safety and efficacy of gene therapy for WAS patients using LVV transduced HSC in combination with reduced intensity conditioning 605 The Infusion of Suicide Gene-Modified Donor T Cells after Hematopoietic Stem Cell Transplantation Prompts Thymic Renewal in Adult Patients by an IL-7 Dependent Mechanism Luca Vago,1,2 Giacomo Oliveira,1 Maddalena Noviello,1 Corrado Soldati,3 Domenico Ghio,3 Immacolata Brigida,4 Alessandro Aiuti,4 Maria Tersa Lupo Stanghellini,2 Jacopo Peccatori,2 Attilio Bondanza,1 Alessandro Del Maschio,3 Claudio Bordignon,5,6 Fabio Ciceri,2 Chiara Bonini.1 Experimental Hematology Unit, San Raffaele Scientific Institute, Milan, Italy; 2Hematology and BMT Unit, San Raffaele Scientific Institute, Milan, Italy; 3Department of Radiology, San Raffaele Scientific Institute, Milan, Italy; 4Pediatric Immunohematology and Bone Marrow Transplant Unit and San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy; 5”Vita-Salute” San Raffaele University, San Raffaele Scientific Institute, Milan, Italy; 6Molmed SpA, Milan, Italy In haploidentical Hematopoietic Stem Cell Transplantation (HSCT), the infusion of donor lymphocytes genetically modified to express the Herpes Simplex Virus Thymidine kinase (HSV-Tk) suicide gene allows GvHD control, while rapidly providing an effective and polyclonal T cell repertoire against pathogens and underlying malignancies (Ciceri and Bonini et al., Lancet Oncology, 2009) In the TK007 phase I/II clinical trial 28 adult patients (median age: 50 years) with hematologic malignancies received purified HSV-Tk-transduced cells after T cell-depleted HSCT, and 22/28 experienced a rapid and stable T cell immune reconstitution Even though their engraftment is necessary to achieve these effects, HSV-Tk+ cells represent the minority of lymphocytes circulating in treated patients Therefore, we hypothesized an indirect role of HSV-Tk+ cells in prompting T cell development from graft progenitors by a thymus-dependent pathway S231 RNA VIRUS VECTORS T cell reconstitution of treated patients demonstrated recovery of naïve HSV-Tkneg T cells The newly reconstituted CD4+ naïve T cells were almost entirely comprised by CD31+ recent thymic emigrants Accordingly, CT scans documented an increase in thymic volume and single joint T cell Receptor Excision Circle counts rose following HSV-Tk cell add-backs Comparison with a cohort of patients subject to T-cell replete HSCT further suggested a unique direct role of HSV-Tk+ cells in promoting thymopoiesis Interestingly, serum levels of IL-7 markedly rose after Tk-cell add-backs, suggesting that the genetically manipulated T cells may mediate the release of this stromal cytokine, in turn supporting the generation and maturation of T cells Notably, in the absence of HSV-Tk cell engraftment, no increase in IL-7 serum levels was observed and patients did non achieve the immune reconstitution The newly generated HSV-Tkneg T cells granted persistent immune competence against infectious agents, which was not compromised in those patients in whom the suicide gene was activated to control GvHD These data show that the infusion of suicide gene-modified T cells induces IL-7 release, boosts the function of the adult thymus and prompts the recovery of a polyclonal, fully competent, T cell repertoire A phase III clinical trial (TK008 study) to assess the efficacy of HSV-Tk+ cells in the context of haploidentical HSCT for leukemia started in Italy, and is currently expanding to multiple centers throughout Europe and US RNA Virus Vectors 606 Assessing the Impact of Lentiviral Vector Integration on Splicing of Cellular Genes at the Genome-Wide Level Stefania Merella,1 Jacopo Sgualdino,1 Daniela Cesana,1 Fabrizio Benedicenti,1 Simone Leo,2 Gianluigi Zanetti,2 Luigi Naldini,1 Eugenio Montini.1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; Center for Advanced Studies, Research, and Development in Sardinia, Pula, Italy Oncogenesis induced by insertional mutagenesis with gene therapy vectors occurs mainly by activation of proto-oncogenes found at or nearby the insertion site This activation often occurs by an enhancer-mediated mechanism or by a process of splicing capture which generates chimeric transcripts comprising portions of vector and cellular mRNAs Although the activation of oncogenes may be reduced by the use of self-inactivating (SIN) design and moderate cellular promoters, how to reduce genotoxic splicing capture events and aberrant transcript formation triggered by vector integration is still unclear We developed a modified Linear Amplification-Mediated (LAM) PCR technique, named cDNA LAM PCR (cLAM-PCR), aimed at retrieving, from the whole transcriptome of LV-transduced cells aberrantly spliced mRNAs that contain lentiviral vector (LV) sequences fused with cellular transcripts in a high-throughput fashion The sequences of cLAM-PCR products were obtained by 454 pyrosequencing and analyzed by a purposely build high-throughput computational pipeline Our pipeline is based on a map-reduce parallelization model, running in a private computer cluster and use a dynamic analysis process composed by different steps implemented as map-reduce applications Thus, chimeric LV-genome sequences are recognized, the nucleotide position of the fused sequence is identified (the splice site), and the remaining portion mapped on the appropriate genome assembly by BLAST Results obtained with different LV constructs show that integrated LVs can perturb the processing of cellular transcripts by interacting with the cellular splicing machinery and fusing with its own splice sites to cellular splice sites both upstream and downstream the integration site So far, 70 different fusion transcripts could be identified in total, 84% of which were fused to known splice sites of gene exons, 6% were fused to uncharacterized cryptic splice sites located in introns and the S232 remaining 10% were fused to genomic sequences not corresponding to any annotated gene We identified several established and previously unknown splice sites within the LV backbone that participate in the aberrant splicing process with variable efficiency Quantitative PCR on different portions of the LV backbone allows measuring the relative contribution to the aberrant splicing process of each LV splice site identified The amount of transcription occurring in regions outside the expression cassette reaches up to the 3% of the entire transgene expression The cLAM-PCR technique, coupled to high-throughput sequencing and the computational power of our specialized data analysis pipeline allows gaining insights into the biology of vector-mediated splicing alteration Since this process could induce neoplastic transformation by the generation of aberrant oncogenic protein, its in-depth characterization is instrumental in the development of next-generation LV with a higher safety profile 607 Read-Through/Splicing-Capture Mechanism Is the Major Determinant of Enhancer Genotoxicity of Vectors with Active LTRs Daniela Cesana,1 Marco Ranzani,1 Cynthia Bartholomae,2 Monica Volpin,1 Stefania Merella,1 Fabrizio Benedicenti,1 Lucia Sergi Sergi,1 Christof von Kalle,2 Manfred Schmidt,2 Luigi Naldini,1 Eugenio Montini.1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; National Center for Tumor Diseases, Heidelberg, Germany We recently developed and validated a new in vivo genotoxicity assay based on systemic vector injection into newborn tumor-prone Cdkn2a-/- mice to address the genotoxic potential of different VSV-G pseudotyped lentiviral vectors (LV) Treatment with an LV with selfinactivating (SIN) Long Terminal Repeats (LTRs) and harboring the strong Spleen Focus Forming Virus (SF) enhancer/promoter sequences in internal position driving GFP expression (SIN.LV.SF GFP), caused a significant acceleration (p