352. Efficient and Non-Toxic Targeting of a Human Safe Harbor Locus with a Lentiviral Vector Associated Meganuclease

RNA VIRAL VECTORS are partially recapitulated with the ML6 chimera and entirely with the ML14, which includes an additional mutation translating in an enhanced targeting specificity by one order of magnitude. Preliminary high throughput integration sites analysis reveals a shift in the histone H3 targeting chimeras profile. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction (Biasco et al, 2011), a feature which might significantly decrease subsequent risk for insertional mutagenesis through undesired enhancer-activation, in combination with a potent and stable genetic insulation, regardless of the integration site inside heterochromatin.

352. Efficient and Non-Toxic Targeting of a Human Safe Harbor Locus with a Lentiviral Vector Associated Meganuclease

Chenxia He,1 Agnès Gouble,2 Alix Bourdel,1 Aleksander Edelman,1 Laurent Poirot,2 Frédéric Paques,2 Olivier Danos.1,3 1 INSERM U845, Université Paris Descartes, Faculté de Médecine Necker, Paris, France; 2Cellectis SA, Paris, France; 3Cancer Institute, University College London, London, United Kingdom. Meganucleases (MN) are site specific endonucleases, with 12 to 45 bp DNA recognition sites. They generate double-strand breaks that can be repaired either by homologous recombination or by non-homologous end joining (NHEJ). MN can be engineered for custom recognition of any genetic locus and used for gene targeting. Our interest is to develop efficient and non-toxic means of targeting transgenes at specific loci for therapeutic purpose. We have previously shown that an active MN could be delivered to cells as a protein associated to a lentiviral vector particle. Here, we further explore the possibilities of associating an I-CreI-derived single chain meganuclease to lentiviral vectors. We have designed a MN, CLS4076, with a unique cutting site on human chromosome 14, at a locus selected as a potential safe harbor for the insertion of therapeutic transgenes. The initial characterization of CLS4076 indicates a high level of activity, associated with significant cell toxicity when introduced by transfection at high doses. Protein fusions were constructed to drive the incorporation of the MN into lentiviral particles. Viral proteins (Vpr and Vpx) and other candidates, all of which interact with HIV-1 Gag, were tested as fusion partners. Different fusions were found to efficiently associate CLS4076 to virions. A quantitative extra-chromosomal assay based on the recombination-mediated rescue of a luciferase reporter gene was used for the initial characterization of MN carrying lentiviral particles. Cell toxicity was measured over 7 days following transduction and found to be absent or minimal at high doses of particles. In comparision, transduction of a regular CLS4076 coding lentiviral vector was associated with up to 45% of cell death. MN transducing virions are being evaluated for targeting the same safe harbor locus on human chromosome 14. Data will be presented on the amount of NHEJ induced at the locus, and on the analysis of homologous recombination in single cell clones.

353. bInSiGHT: Bioinformatics Integration Sites Tool for Gene Therapy with High-Throughput Platforms

Andrea Calabria,1 Fabrizio Benedicenti,1 Davide Cittaro,2 Elia Stupka,2 Christof von Kalle,3 Manfred Schmidt,4 Luigi Naldini,1 Eugenio Montini.1 1 San Raffaele Telethon Institute for Gene Therapy, Milano, Italy; 2 Center for Translational Genomics and Bioinformatics, San Raffaele, Milano, Italy; 3National Center for Tumor Diseases, Heidelberg, Germany.

Insertional mutagenesis is one of the major hurdles of gene therapy with integrating vectors. Analysis of chromosomal vector integration S138

sites in vector marked cells from gene therapy patients and preclinical models has enabled to detect in vivo selection of gain-of-function insertional mutants even before they progress to overt malignancy. For this reason, over the last years there has been a constant increase in the amount of sequencing and mapping of vector/genomic DNA junctions and statistical tools to improve biological investigations and their interpretation. Recently, Next Generation Sequencing (NGS) approaches have been exploited in Gene Therapy to greatly enhance the analytical power of integration site analysis. However, NGS analytical advances are balanced by computational drawbacks such as the flooding of data that needs to be carefully managed and processed with smart pipelines on distributed high-performances infrastructures. Thus NGS-structured and standardized pipelines for biologists are strongly required. We developed a NGS bioinformatics pipeline, both for Illumina and Roche platforms, for integration sites analysis with intuitive web-based interfaces, enabling quality controls and highperformance properties: bInSiGHT. We designed bInSiGHT data processing activities grouped in: (a) sequence reads quality control, avoiding unreliable data analysis, (b) raw data cleaning and vector sequences trimming, (c) sequence reads grouping and clustering, to estimate the number of integration sites before mapping sequences, (d) reads mapping to reference genome (e) post-processing data filtering and refinement of integration sites for subsequent analyses (i.e. integration sites association to annotated genomic features, common integration site identification and complexity estimation as surrogate of clonal diversity of vector marked cells). We developed and tested the system on cluster environment. We successfully validated bInSiGHT characterizing the integration profile on 3 patients enrolled in a lentiviral vector-based hematopoietic stem cell gene therapy trial for metachromatic leukodystrophy (MLD) performed in our institute. Exploiting both optimized alignment methods and parallelization algorithms, we obtained a speedup of 20x when compared to our previously developed version of data analysis pipeline. Moreover, the identification and quantification of genuine integration sites based on clustering of vector integrations without the need of genomic mapping has increased the numbers of integrations that can be tracked during time and between different hematopoietic lineages. By the use of bInSiGHT we found that MLD treated patients have a large repertoire of vector integrations suggesting high levels of polyclonal reconstitution. Moreover, no significant clonal expansions were detected and no skewing towards CIS or cancer genes during time suggesting lack of genotoxic events at one year after transplantation.

354. The Integration Profile of Lentiviral Vector in CD34+ Hematopoietic Stem Cells Is Dependent on the Cell Cycle Status of the Target Cell

Eleni Papanikolaou,1,2 Ekati Drakopoulou,1,2 Evangelos Stamateris,1,2 Alexandros Polyzos,3 Anna Paruzynski,4 Cynthia Bartholomae,4 Manfred Schmidt,4 Christof von Kalle,4 Nicholas P. Anagnou.1,2 1 Laboratory of Biology, University of Athens School of Medicine, Athens, Greece; 2Laboratory of Cell and Gene Therapy, Biomedical Research Foundation of the Academy of Athens, Athens, Greece; 3Deparment of Molecular Biology, Biomedical Research Foundation of the Academy of Athens, Athens, Greece; 4 National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany. Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may activate proto-oncogenes via viral integration, a phenomenon called insertional mutagenesis. Although, such risks had been originally estimated as extremely low, the report of leukemia due to insertional activation of the LMO2 gene following gene therapy for X-SCID in a minority of patients, led to a Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy