607 Read Through/Splicing Capture Mechanism Is the Major Determinant of Enhancer Genotoxicity of Vectors with Active LTRs Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American S[.]
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