354 The Integration Profile of Lentiviral Vector in CD34+ Hematopoietic Stem Cells Is Dependent on the Cell Cycle Status of the Target Cell Molecular Therapy Volume 20, Supplement 1, May 2012 Copyrigh[.]
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 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 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 San Raffaele Telethon Institute for Gene Therapy, Milano, Italy; 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 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 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; 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 RNA VIRAL VECTORS re-evaluation of the mechanisms operating in insertional mutagenesis To this end, we sought to determine the viral integration pattern based on the cycle stage of the target cell For that reason, cord blood CD34+ cells were transduced with a GFP-LV after synchronization at the G1 phase (G1 population) by addition of thymidine, or after arrest at the G1/S phase (G1/S population) by addition of aphidicoline or at mitosis by addition of nocodazole or taxol (MN or MT populations respectively) Overall, we mapped 197 integration sites (IS), i.e 49 in the untreated cells, 73 in the G1 population, 12 in G1/S population and 53 and 10 in the MN or MT populations, respectively These results show that taxol and aphidicoline severely inhibit viral integration Relative to the integration pattern, the percentage of the “targeted” cancer-correlated genes was significantly lower in the thymine population (4% vs 34% in the untreated cells), while we observed an increased probability (1%) of viral integration in the SPATS2 and OR13H1 genes These novel data provide the impetus for preclinical in vitro models for the engineered cells, leading eventually to patients’ safety 355 Generation of a Barcoded Lentiviral Plasmid Library for Analyzing Clonal Complexity and Evaluation of Library Complexity Using Next Generation Sequencing Claire T Deakin,1 Samantha L Ginn,1 Claus V Hallwirth,1 Ian E Alexander.1,2 Gene Therapy Research Unit, Children’s Medical Research Institute and Children’s Hospital at Westmead, Australia; Discipline of Paediatrics and Child Health, University of Sydney, Australia Successful clinical trials of gene therapy targeting the hematopoietic compartment have demonstrated retroviral gene delivery to relatively small numbers of hematopoietic progenitors can cure a disease phenotype However, little is known about how many progenitor clones contribute to differentiation of different hematopoietic lineages nor the long-term dynamics of differentiation Such knowledge may help define optimal cell doses in clinical applications Furthermore, the development of leukemia in five infants treated in gene therapy trials for X-linked severe combined immunodeficiency highlighted the need for sensitive methods to detect malignancies in clinical applications using integrating vectors Existing methods based on integration site analysis are associated with biases introduced by the use of restriction endonucleases leading to preferential amplification of smaller amplicons The development of barcoded lentiviral vectors may enable individual cells to be uniquely tagged during transduction and subsequently monitored within a mixed population in an unbiased fashion Analyzing such samples requires accurate identification of large numbers of unique barcodes present at low frequency and in the order of thousands to millions of combinations Developments in next generation sequencing (NGS) have opened the potential for such analysis A barcoded lentiviral plasmid library was generated by inserting a short sequence containing degenerate nucleotides into a lentiviral construct containing the elongation factor 1α (EF1α) promoter and the interleukin-2 receptor common gamma chain (γc) Preliminary analysis of library complexity was conducted using an Illumina HiSeq2000, however, analysis was confounded by the instrument’s high error rate under the conditions used and the absence of a reference sequence for a complex library where the sequence of each barcode was unknown Analysis of error rates revealed the most accurate region of the Illumina read was within the first 35 bases, such that sequencing of the barcode region, located after position 40, was impugned by instrument error Furthermore, a 33-base stretch of homogeneous sequence at the front-end of the read did not favor the platform’s algorithms for registering individual nucleotide clusters and calculating phasing and dephasing parameters associated with error The barcode insert was redesigned in a platform-specific manner Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy to locate the barcode at the front-end of a sequence read and maximize use of the highest quality Illumina sequence data A second barcoded EF1α.γc lentiviral plasmid library specific for the Illumina platform was produced and is currently undergoing analysis with the inclusion of an internal standard curve of barcode complexity Collectively, these measures should allow us to define the utility of the Illumina platform for this type of analysis This study has shown careful design based on an understanding of sequencing technology is required when NGS is used to analyze the complexity of barcoded lentiviral vectors 356 Codon Optimization of RD114-TR Envelope Glycoprotein Leads to Lack of Correct Processing and Therefore Correct Function of the Envelope Eleonora Zucchelli,1 Anna Stornaiuolo,1 Sergio Bossi,1 Claudio Bordignon,1 Gian Paolo Rizzardi,1 Chiara Bovolenta.1 MolMed S.p.A., Milano, Italy MolMed S.p.A has leading expertise in cell and gene therapy product development, with an in-house cGMP facility authorized for the production and release of clinical-grade medicinal products for human use Several years ago we initiated a long-term development plan to generate packaging cell lines for semi-stable and stable production of HIV based lentiviral vector (LV) for clinical application Our strategy is based on the sequential integration of the constitutively expressed viral genes (gag, pol, rev and the feline endogenous retroviral RD114-TR env) through viral vector transduction Packaging genes have been inserted by means of an AAV Rep78mediated baculo-AAV hybrid vector obtaining an intermediate cell clone, named PK-7, whereas the env gene has been integrated through SIN-LV-specific delivery Although the titer of the RD114TR pseudotyped LV resulting upon transient transfection of SIN-LV is in the range of those reported in literature (1 × 105 TU/ml), and that of LTR-LV upon stable integration is even higher (1 × 106 TU/ ml), we sought to increase LV efficiency by codon optimization of RD114-TR gene To this aim, we initially codon optimized (co) the entire RD114-TR ORF, RD114-TRco (GENEART, Germany) and fully characterized the new product by assessing its expression and function by Western blot analysis and RD114-TRco pseudotyped LV titer determination, respectively We used two specific anti-RD114TR Abs, the anti-SU and anti-TM, both recognizing the precursor (PR) besides the surface (SU) and trans-membrane (TM) specific subunits In fact, RD114-TR is normally translated as a 564-aa PR, that is processed by the membrane-associated endoprotease furin in 361-aa SU and 203-aa TM subunits The migration of RD114-TR molecules in a SDS-PAGE depend on their glycosylation status, that is, the glycosylated forms of both PR and SU migrate at 75 kDa, whereas, after deglycosylation, the PR at 60 kDa and the SU at 40 kDa, and the glycosylated and deglycosylated TM at ca 18 kDa and ca 15 kDa, respectively Remarkably, we found that RD114-TRco was transcribed and its PR abundantly translated, but not processed Western blot analysis in fact revealed that in the glycosylated and deglycosylated conditions, respectively, the 75-kDa and 60-kDa bands were normally present, but not the 18-kDa and 15-kDa bands As a consequence the titer of the corresponding RD114-TRco pseudotyped LV was negative Next, we generated two half-recoded proteins, the RD114-TR-5’co and RD114-TR-3’co, by codon optimizing only the 5’ half and the 3’ half of the ORF, hypothesizing that partial recoding could eliminate the negative effect likely generated by silent mutation(s) introduced during recoding causing either abnormal speed of the transcription/translation process and/or abnormal folding of the protein, which could eventually prevent furin cleavage Unfortunately, also the two half-optimized envelopes were not functional and their overall maturation was severely impaired In conclusion, our studies suggest that the RD114-TR envelope glycoprotein is not suitable for codon optimization and this strategy cannot be applied to improve its performance S139 ... VIRAL VECTORS re-evaluation of the mechanisms operating in insertional mutagenesis To this end, we sought to determine the viral integration pattern based on the cycle stage of the target cell. .. malignancies in clinical applications using integrating vectors Existing methods based on integration site analysis are associated with biases introduced by the use of restriction endonucleases leading... containing degenerate nucleotides into a lentiviral construct containing the elongation factor 1α (EF1α) promoter and the interleukin-2 receptor common gamma chain (γc) Preliminary analysis of library