85 Modeling the Genomic Integration Site Distribution of Genotoxic and Non Genotoxic Retroviral Vectors in Hematopoietic Stem/Progenitor Cells In Vivo Molecular Therapy Volume 17, Supplement 1, May 20[.]
RNA VIRUS VECTORS I 83 Optimization of Lentiviral Transduction of Human Peripheral Blood Lymphocytes for TCR Adoptive Cell Therapy Neel K Karne,1 Scicheng Yang,1 Stephanie G Downey,1 Steven A Rosenberg,1 Richard A Morgan,1 Steven A Feldman.1 Department of Surgery, National Cancer Institute, National Institutes of Health, Bethesda, MD Adoptive cell therapy using tumor infiltrating lymphocytes (TIL) for the treatment of patients with metastatic melanoma has shown dramatic tumor regressions As an alternative to TIL, peripheral blood lymphocytes (PBL) have been genetically modified by γ-retroviral transduction to introduce a tumor-specific T cell receptor (TCR) followed by a rapid expansion Lentiviral vectors can effectively transduce PBL at much higher cell densities, theoretically obviating the need for rapid expansion and may enable the adoptive transfer of a less differentiated cell population The goal of this study was to determine optimal parameters for transduction and expansion PBL using lentiviral vectors After development of an optimized lentiviral vector encoding a MART-1-specific TCR, we explored cell density at transduction, time of transduction, stimulation method, and various interleukin (IL-2) concentrations post-transduction Increasing cell density did not affect transduction efficiency, but did result in decreased cell expansion Anti-CD3/anti-CD28 bead stimulation of PBL resulted in a higher percentage of transduced T cells which expanded approximately 2-fold better when compared to OKT3-stimulated cells Interestingly, transduced cells stimulated with OKT3 preferentially expanded the CD8+ T cell subset Functionally, there was no difference between cells stimulated with beads or OKT3 Increased concentrations of IL-2 or a combination of soluble OKT3 and anti-CD28 did not affect transduction or subsequent cell expansion; however, inclusion of soluble OKT3 and/or anti-CD28 post-transduction resulted in higher numbers of CD8+ T cells as compared to bead or OKT3 stimulation alone These findings provide a strategy for the optimization of lentiviral transduction of PBL with the goal of developing a clinical protocol for the treatment of patients with metastatic melanoma 84 Histone H3.1 Enhances the Retroviral Transduction of a Hematopoietic Cell Line Model and Primary Hematopoietic Stem Cells Pascal R Beauchesne,1,2 Katherine J Bruce,1,2 Michelle Miller,3 Sanja Sekulovic,3 Bruce D Bowen,2 R Keith Humphries,3 James M Piret.1,2 Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada; 2Chemical and Biological Engineering, University of British Columbia, Vancouver, BC, Canada; 3Terry Fox Laboratory, British Columbia Cancer Research Center, Vancouver, BC, Canada; 4Medicine, University of British Columbia, Vancouver, BC, Canada The effectiveness of recombinant retrovirus-mediated gene transfer can be severely limited due to mass transport limitations, thereby requiring vector concentration steps and repeated exposures of the target cells to achieve desirable scientific and therapeutic outcomes The low diffusivity (∼6 x 10-8 cm2/s) and short half-life (∼6 h) of retroviruses combined with various physico-chemical forces limit the encounter frequency of vectors and target cells We previously reported that whole cell or nuclei lysate fractions increased the transduction of TF-1 cells by over 40-fold relative to a no additive control Despite their effectiveness, the complexity of cell lysates may limit their use, especially at the clinical level Defined cationic reagents such as protamine sulfate, an arginine-rich peptide, have been shown to improve retrovirus-mediated gene transfer by 5- to 8-fold in TF-1 cells Histones, a group of cationic nuclear proteins rich in lysine and arginine residues, provide comparable enhancement S34 However, their use with retroviral vectors has been limited to mixed tissue-derived preparations, thus confounding the properties of the individual histone types To further define the active components within the nuclear lysate fraction, we screened the effect of individual histone types on the transduction efficiency of TF-1 cells A lysinerich fraction (f1) predominantly composed of histone H1 and an arginine-rich fraction (f3) mainly composed of histone H3 extracted from calf thymus were added to TF-1 cells and retroviral vectors with a GFP reporter gene produced from PG-13 packaging cells Unlike protamine sulfate that requires a tissue culture-treated surface, both histone fractions enhanced retroviral transduction independently of surface type While the f1 histone fraction yielded a similar increase as protamine sulfate, the f3 fraction enhanced transduction by over 35-fold, a 4-fold improvement over protamine sulfate Similar results were obtained using unmodified human recombinant histones as H1, H2A, H2B and H4 matched the effect of protamine sulfate, while the H3.1 variant provided a 35-fold increase in transduction This recombinant H3.1 histone protocol was successfully applied to the retroviral transduction of mouse bone marrow enriched in hematopoietic stem cells (HSC) by 5-FU pre-treatment and compared to a co-culture method The addition of histone H3.1 was as effective for transducing the HSC population where 50% of donor-derived cells were GFP+ compared to 43% using the co-culture method and 5.6% with no additives as verified by a long term reconstitution assay This novel protocol based on the recombinant human histone H3.1 provides a fully-defined alternative with higher transduction efficiency and is compatible with sensitive primary cell populations such as HSCs 85 Modeling the Genomic Integration Site Distribution of Genotoxic and Non Genotoxic Retroviral Vectors in Hematopoietic Stem/ Progenitor Cells In Vivo Eugenio Montini*,1 Daniela Cesana*,1,2 Jacopo Sgualdino,1,2 Manfred Schmidt,3 Alessia Capotondo,1,2 Francesca Sanvito,4 Cynthia Bartholomä,3 Marco Ranzani,1,2 Fabrizio Benedicenti,1 Lucia Sergi Sergi,1 Claudio Doglioni,4 Alessandra Biffi,1 Christof VonKalle,3 Luigi Naldini.1,2 HSR-TIGET, Milan, Italy; 2San Raffaele University, Milan, Italy; NTC, Heidelberg, Germany; 4HSR, Milan, Italy Gamma-Retroviral vectors (gRV), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis We previously dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-treated tumor prone mouse hematopoietic stem/progenitor cells (HSPC) By swapping genetic elements between gRV and lentiviral vectors (LV), we demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs, and that self-inactivating (SIN) LTRs enhanced the safety of gRVs By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as gRV This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by gRVs This integration-site bias was intrinsic to gRVs, as it was also observed for SIN-gRVs that lacked genotoxicity in our model These studies validate the biosafety of the SIN design and the lower impact of LV ISS on genotoxicity The use of a modified LV with active LTR was instrumental to dissect the role of ISS in modulating vector genotoxicity in mice Here we took advantage of this genotoxic LV to mutagenize human CD34+ HSPC and study its integration pattern in vitro and in vivo in human/mouse hematochimeras Cord blood CD34+ cells were transduced with our genotoxic or the standard SIN LV and transplanted into Rag2/gchain-/- mice to allow expansion and differentiation in vivo High Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy BIOLOGY OF AAV AND VECTOR DEVELOPMENT I levels of transduction were achieved in vitro (>90%) and substantial engraftment of marked human cells was found in the bone marrow, spleen and thymus of mice at 13 weeks after transplant The genomic integration profile of the these vectors is currently being determined after 2, 4, and weeks of in vitro culture and in blood and organs of transplanted mice at 13 and 20 weeks after transplant Comparison of the genomic integration pattern of the genotoxic and SIN LV at different time points will indicate whether significant differences can be observed between these vectors and during time We expect that the genotoxic LV induces clonal dominance driven by insertional mutagenesis and that its integration profile preferentially target specific gene classes in vivo and uncover new genes whose deregulation may lead to clonal outgrowth of human hematopoietic cells Moreover, the genotoxic LV integration profile could be used as reference against which to assess integration data obtained from the growing number of clinical trials using LV for HSPC gene therapy *equal contribution 86 Functional Analysis of Retroviral Vector Integration Sites in the Treatment of Murine X-Linked Chronic Granulomatous Disease Using Gene Ontology Troy B Hawkins,1 Mohammed A Sadat,2 Jessica Dantzer,1,3 Ken Cornetta,1,2,4 Sean D Mooney,1,3 Mary C Dinauer.1,2,5 Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN; 2Pediatrics, Wells Center for Pediatric Research, Riley Hospital for Children, Indianapolis, IN; 3Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN; 4Medicine, Indiana University School of Medicine, Indianapolis, IN; 5Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN Retroviral vectors have become a common and effective tool in the treatment of the genetic defects characteristic of inherited immune disorders such as X-linked Severe Combined Immunodeficiency (SCID) and Chronic granulomatous disease (CGD) This treatment involves the genetic modification of hematopoietic stem cells (HSCs) by random integration of retroviral DNA into the host cell genome followed by competitive clonal selection and engraftment Gene therapy by transduction of retroviral vectors has been shown to effect unintended consequences on host cells, disrupting cellular gene function through activation by potent viral enhancers located in vector long-terminal repeats (LTRs) Here we describe analysis of Gene Ontology (GO) functional annotations of genes neighboring 236 retroviral insertion sites (RISs) in a study of gene therapy treatment of murine X-CGD Genomic regions downstream of vector integrations were amplified and identified by ligation-mediated PCR (LM-PCR), sequenced, and aligned to corresponding regions in the mouse genome Genes within ± 300kb were identified by the SeqMap tool and annotated with GO biological process and molecular function terms as provided by MGI Gene annotation subgraphs were completed using the full set of ancestral terms and analyzed for overrepresentation against the MGI background using the hypergeometric distribution Terms identified as over-represented (phg ≤ 0.05) were compared between test cohorts in the study: lethally vs sub-lethally irradiated mice and primary vs secondary and tertiary transplant recipients Integration-associated genes in both the lethally and sublethally irradiated cohorts were enriched for biological process and molecular function terms related to transcription Some differences between the two groups were identified, with genes near RISs in lethally irradiated mice being significantly more enriched (pt ≤ 0.05) for cell proliferation, GTPase signal transduction, and hematopoiesis, and those in sub-lethally irradiated mice being more enriched for phosphorylation and protein modification Among specific tissue subsets, genes near integrations in spleen samples were enriched for Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy phosphate metabolism and kinase activity in sub-lethally irradiated mice and those in bone marrow (BM) or both BM and spleen were enriched for regulatory processes and transcription factor activity in lethally irradiated mice Secondary and tertiary transplant recipients were enriched for genes related to transcription, similar to those found in primary transplant recipients Biology of AAV and Vector Development I 87 The Viral Capsid Significantly Influences the Pathway and Rate of Adeno-Associated Virus Intracellular Trafficking in HeLa Cells Nicholas W Keiser,1 Paul M Kaminsky,1 Ziying Yan,1 John F Engelhardt.1 Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA Intracellular trafficking has been identified as a rate-limiting step in adeno-associated virus (AAV) transduction Following receptor binding, AAV enters the cell via clathrin-mediated endocytosis, and traffics through various endosomal compartments before it escapes into the cytoplasm and enters the nucleus Previous studies by our laboratory have identified the late and recycling endosomes as vesicular compartments through which AAV type (AAV2) traffics prior to nuclear entry in HeLa cells However, the precise endosomal trafficking pathway of other AAV serotypes is not as well established We hypothesized that differences in capsid structure would result in endocytic trafficking pathways for AAV type and that were fundamentally distinct from that of AAV2 In order to compare endocytosis and trafficking pathways of AAV1 and AAV5 to AAV2, we labeled these AAV serotypes with different fluorochromes, allowing for simultaneous imaging of two different serotypes in cells HeLa cells were co-infected with Alexa 488-labeled AAV1 or AAV5 along with Alexa 568-labeled AAV2 Cells were fixed at various time points up to 60 minutes post-infection and analyzed by confocal fluorescence microscopy Co-localization analysis showed that the most overlap between AAV2 and AAV5 fluorescence occurred during the first 20 minutes of infection, while the trafficking pathways diverged at later time points In contrast, there was minimal overlap between AAV1 and AAV2 during both early and late stages of infection In addition, we noted stark differences in the rate of AAV trafficking from the plasma membrane to the nucleus AAV1 entered cells within the first 5-10 minutes of infection and rapidly trafficked to the perinuclear space, whereas AAV2 and AAV5 entered more gradually, and did not accumulate in the perinuclear space until 60 minutes post-infection Using quantitative PCR, we compared the number of viral genomes in the nucleus and cytoplasm following infection Consistent with the results from confocal imaging suggesting rapid trafficking to the nucleus for AAV1, we found that a greater percentage of AAV1 genomes were present in the nucleus compared to the other two serotypes However, we found that at the same multiplicity of infection (MOI), the number of AAV1 genomes endocytosed into Hela cells was significantly less than that for AAV2 or AAV5 We also noted significantly lower levels of transduction with AAV1 compared to AAV2 and AAV5 These findings show that barriers to viral entry, intracellular trafficking, and transgene expression may be different for each AAV serotype In conclusion, the results of this study suggest that distinct intracellular trafficking pathways for AAV1, 2, and exist in HeLa cells We propose a model by which the utilization of different receptors determines the specific pathway and rate of AAV trafficking to the nucleus Understanding the determinants in the viral capsid that direct viral movement through various steps in the infectious process may enable construction of virions that more effectively express their encoded transgenes S35 ... transplant The genomic integration profile of the these vectors is currently being determined after 2, 4, and weeks of in vitro culture and in blood and organs of transplanted mice at 13 and 20 weeks... Comparison of the genomic integration pattern of the genotoxic and SIN LV at different time points will indicate whether significant differences can be observed between these vectors and during time... outgrowth of human hematopoietic cells Moreover, the genotoxic LV integration profile could be used as reference against which to assess integration data obtained from the growing number of clinical