88 site specific genome modification of human primary t lymphocytes to improve the safety and efficacy of adoptive immunotherapy

88 Site Specific Genome Modification of Human Primary T Lymphocytes To Improve the Safety and Efficacy of Adoptive Immunotherapy Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The Ame[.]

TARGETING GENE MODIFICATION AND INTEGRATION 86 The Effect of Surface Modication of Adenovirus with an Arginine-Grafted Bioreducible Polymer on Transduction Efciency and Immunogenicity in Cancer Gene Therapy Pyung-Hwan Kim,1,2 Tae-il Kim,3 James W Yockman,3 Sung Wan Kim,3,4 Chae-Ok Yun.1,2 Graduate Program for Nanomedical Science and Technology, Yonsei University, Seoul, Republic of Korea; 2Brain Korea 21 Project for Medical Sciences and Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea; Department of Pharmaceutics and Pharmaceutical Chemistry, Center for Controlled Chemical Delivery, University of Utah, Salt Lake City, UT; 4Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea Adenoviral vectors have been widely used for cancer gene therapy due to many advantages, such as high transduction efciency, but safety concerns related to severe immunogenicity and other side effects have led to careful reconsideration of their use in human clinical trials To overcome these issues, a strategy of generating hybrid vectors that combine viral and non-viral elements as more intelligent gene carriers has been employed Here, we coated adenovirus (Ad) with a novel arginine-grafted bioreducible polymer (ABP) via electrostatic interaction We examined the transduction effect of ABP-coated Ad complex at various ABP molecules/ Ad particle ratios We also examined the coating of Ad with ABP polymers at the optimal polymer ratio using dynamic light scattering and transmission electron microscopy and conrmed the coating by NaCl treatment because the slat ions of NaCl is more strong ion interaction than that of ABP to virus The characterization of complex was compared the size and surface charge of ABP-coated Ad complex formed by physical interaction compared with naked Ad by Zetasizer In both high and low coxsackie virus and adenovirus receptor (CAR)expressing cells, ABP-coated Ad complex produced higher levels of transgene expression than cationic polymer 25K PEI Notably, high cytotoxicity was observed with 25K PEI-coated Ad complex treatment In addition, ABP-coated Ad complex was not signicantly inhibited by serum, in contrast to naked Ad Moreover, ABP-coated Ad complex signicantly reduced the innate immune response relative to naked Ad, as assessed by interleukin-6 (IL-6) cytokine release from macrophage cells Overall, our studies demonstrate that the combination of Ad with ABP polymer offers the potential to increase the efciency of vectors for gene therapy by shielding the virus from deactivation by the immune system, and may make systemic administration feasible Targeting Gene Modication and Integration 87 Safe and Efcient Delivery of Meganucleases for Lentivirus-Mediated Gene Targeting Araksya Izmiryan,1 Alix Bourdel,1,2 Arnaud Jollet,1 Frédéric Pâques,2 Olivier Danos.1 Université Paris Descartes, INSERM U781, Paris, France; Cellectis SA, Romainville, France Gene therapy treatments using retroviral and lentiviral vectors have revealed the genotoxicity associated with random insertion of vector DNA into the genome Our goal is to design lentiviral vectors with which a therapeutic sequence is inserted at a universal safe locus in the human genome, using homologous recombination (HR) Lentiviralmediated HR occurs at high frequency when a DNA recombination matrix is delivered to cells along with a site specic endonuclease that creates a locus-specic double strand break Heterodimeric Zinc nger Nucleases as well as single chain meganucleases can be used for this purpose However, for therapeutic applications, it is preferable to have S34 an appropriate control of the amount of endonuclease in the target cells, and especially, to avoid sustained expression One possibility is to vectorize the protein itself, instead of an expression cassette Here we describe experiments showing that highly efcient HR is obtained when the nuclease is delivered as a protein incorporated in the lentiviral particles I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein Non-integrating lentiviral vectors (NILV) containing a recombination matrix and the I-SceI fusion protein were produced at high titers We have used a CHO-derived reporter cell line in which I-SceI mediated HR events result in the repair of a puromycin resistance gene These cells were transduced by NILVs containing both I-SceI as a Vpr fusion and the recombination matrix, or by separate vectors encoding either the recombination matrix or I-SceI Puromycin resistant cell clones were selected and analysed by PCR and Southern blot for targeting events A majority of puromycin resistant clones contained the expected genomic structure and globally, our data indicate that the transfer of the endonuclease as a virion incorporated protein results in up to 5% of HR events, in a dose dependent manner HR levels were lower when the fusion protein and the recombination matrix were provided in separate viral particles, suggesting that the physical association of the two in the same lentiviral pre-integration complex may be important for efcient targeting Interestingly, the levels of HR obtained when I-SceI was encoded by the lentiviral vector were consistently lower Finally, we have shown that Vpr fusions could also be used for the transduction of a single chain meganuclease (scMN) engineered from I-CreI with a recognition site in the human Rag-1 gene We are now using this system with various scMN designed to target transgene insertion in candidate “safe havens” on the human genome 88 Site-Specic Genome Modication of Human Primary T Lymphocytes To Improve the Safety and Efcacy of Adoptive Immunotherapy Pietro Genovese,1,2 Elena Provasi,3,2 Angelo Lombardo,1,2 Zulma Magnani,3 Andreas Reik,5 Pei-Qi Liu,5 Oscar Muniz Pello,1 Jurgen Kuball,4 Philip D Gregory,5 Michael C Holmes,5 Philip D Greenberg,4 Chiara Bonini,3 Luigi Naldini.1,2 HSR-TIGET, Milan, Italy; 2San Raffaele University, Milan, Italy; San Raffaele Scientic Institute, Milan, Italy; 4Fred Hutchinson Cancer Research Center, Seattle; 5Sangamo BioSciences, Richmond, CA Editing the genetic content of clinically relevant primary cells, such as T lymphocytes, has long been a major goal for gene therapy Indeed, development of an efcient method for gene targeting in these cells can overcome many of the technological and safety issues of the current gene replacement strategies for adoptive immunotherapy Here, we present a novel approach based on Zinc Finger Nucleases (ZFN) and Integrase Defective Lentiviral Vectors (IDLV) for: i) targeted integration of transgenes and ii) site-specic gene knock out in primary human T cells First, we developed a platform for site-specic integration into two putative safe genomic harbors, the CCR5 gene and the AAVS1 locus Codelivery of ZFNs targeting either site by Adenoviral vector (Ad5/F35) in association with IDLV containing a GFP expression cassette anked by locus homology arms, resulted in efcient site-specic integration (6%) Molecular analysis on GFP+ clones conrmed site-specic integrations at the ZFN targeted sites with disruption of the sister allele by NHEJ in up to 87% of clones GFP expression was higher from the AAVS1 than the CCR5 site Moreover, gene expression analyses showed that integration of an expression cassette into AAVS1 did not perturb expression of anking genes To test the feasibility of this approach in a therapeutically relevant setting, we transferred exogenous T cell receptor (TCR) genes ZFN-mediated integration of a high-avidity TCR specic for the WT1 tumor antigen in TCR βneg Jurkat cells Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy TARGETING GENE MODIFICATION AND INTEGRATION resulted in ∼18% gene targeting Next, exploiting ZFNs to create gene knockouts we addressed a major hurdle with TCR gene transfer, the co-expression of endogenous and tumor specic TCRs in the same cell The expression of TCR chains not only diminishes expression of the desired TCR, due to competition for CD3 molecules, but also leads to TCR mispairing, which results in unpredictable specicities that can be autoreactive Thus, we developed ZFNs specic for the constant regions of TCR β chain gene (TRBC1 and TRBC2) and exploited the NHEJ-repair to disrupt the endogenous TCR ZFN treatment resulted in functional inactivation of this gene in up to 7% of primary T cells, as indicated by generation of cells that not express the CD3/TCR complex on cell surface, in the absence of evident toxicity Molecular analyses revealed equal NHEJ mediated gene disruption of both TRBC loci LV-mediated transfer of the WT1-TCR was efciently achieved in CD3neg sorted cells (>45%) and resulted in higher expression of the tumor specic TCR than that observed with conventional gene transfer Overall, our results demonstrate that both ZFNs mediated gene addition and gene disruption can be successfully combined in T lymphocytes to facilitate rapid generation of effective and safe T cells for adoptive immunotherapy *equal contribution 89 Single-Strand Nicks Induce Homologous Recombination with Less Toxicity Than DoubleStrand Breaks Using an AAV Template Michael J Metzger,1 Audrey McConnell-Smith,2 Barry L Stoddard,2 A Dusty Miller.1 Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA The enhancement of homologous recombination (HR) using targeted double-strand breaks (DSBs) has the potential to correct gene defects in their endogenous loci, avoiding many problems which have plagued traditional gene therapy However, even a perfectly site-specic DSB is a DNA damaging event, and resulting toxicity and potential for mutagenesis and translocations are serious problems for this strategy We compared the ability of nicks and DSBs to enhance homologous recombination using the homing endonuclease I-AniI and a redesigned variant which produces only single-strand nicks When both template and I-AniI expression plasmids were transfected into 293 cells containing an integrated copy of an inactive lacZ target, we showed that the nickase enhances HR up to 300-fold above transfection of template plasmid alone (compared to 8,000-fold enhancement with the DSB-inducing enzyme) When we delivered the template with an AAV vector and the endonuclease construct with a lentivirus for longer expression, we also found that both DSBs and nicks enhance HR in a manner dependent on the amount of endonuclease used While HR was induced with lower amounts of DSB-inducing enzyme than with nick-inducing enzyme, the toxicity observed with the DSB-inducing enzyme was far more severe (>80% cell death at an MOI of 1) In contrast, the toxicity of the nicking endonuclease was low and was not distinguishable from the toxicity of an inactive endonuclease or the toxicity of an empty lentivirus vector expressing only the mCherry marker used to titer vectors Due to this DSB toxicity, the maximum amount of HR observed with nicks and with DSBs was similar (20-60 nick-induced foci compared to 50-80 DSB-induced foci in 5x104 cells) For both assays, two different lacZ target sites were investigated: one in which the 19 bp I-AniI recognition site replaced a 19 bp region in the lacZ gene and the other in which the I-AniI site was inserted into the same location Interestingly, the lacZ target with the “replacement” inactivating mutation supported nick-induced HR at a 10-fold higher rate than the target with the “insertion” mutation in both the AAV assay and the transfection assay, while no difference in DSB-induced HR was observed between the two targets It has been suggested that nicks could only stimulate HR upon conversion to a DSB, making nicks Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy simply a less efcient version of DSB-induced HR; however, these results suggest that is incorrect Both the observation that nicks can stimulate HR with lower toxicity than DSBs and the observation that target site design effects nick-induced HR but not DSB-induced HR strongly argue that nicks induce HR through a different mechanism than DSBs Therefore, this strategy of HR with a nicking enzyme and a viral delivery system may be useful in clinical gene therapy applications, allowing for more efcient gene correction without the toxicity and mutagenic activity of DSBs 90 Targeted Gene Insertion in Human Chromosomes Using a Homing Endonuclease Byoung Y Ryu,1,2 Michael T Certo,1,2 Mikhail Garibov,1,2 Andrew M Scharenberg,1,2 David J Rawlings.1,2 Northwest Genome Engineering Consortium (NGEC), Seattle, WA; 2Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA Homing endonucleases (HE) are a class of rare cutting nuclease that recognize and cleave 19-22 base pair (bp) sequences with a limited degree of degeneracy They represent an emerging tool for genome engineering as they can be used to induce site specic DNA double strand breaks (DSB) and initiate homologous recombination or gene insertion Among known HE, I-AniI recognizes 19 bp asymmetric sequences, and has no exact match in human genomic sequence databases To determine if I-AniI can induce DSB in cells, we performed BLAST search to the known I-AniI target site to identify near-cognate matches in the human genome potentially susceptible to cleavage A set of 60 near-cognate sites were individually cloned into reporter vectors in order to compare rates of in cellulo cleavage to levels of cleavage predicted via in vitro proling Three near-cognate sites were identied that cleaved with high efciency in the reporter assay including: one intronic, one exonic, and one intergenic region site To evaluate the accessibility of these sites within genomic DNA, integration decient lentiviral vectors (IDLV) were developed to express I-AniI variants in human pre-B cell line, Nalm-6 cells As DSB are frequently repaired through non-homologous end joining (NHEJ) pathway, the rate of genomic NHEJ events after IDLV transduction was used as surrogate marker for cleavage efciency Consistent with in cellulo cleavage, all three near-cognate sites exhibited similar NHEJ rates ranging from 15 to 20% The percentage of NHEJ events was even higher (>70%) when integrating LV were used to stably express I-AniI in human cells supporting the conclusion that I-AniI can recognize and cleave these sites in vivo The intergenic locus was also evaluated as a site for targeted gene insertion When a GFP expression cassette (1.8 kb) with anking homologous sequences was introduced into human Nalm-6 cells along with I-AniI, an estimated 1% of targeted GFP gene insertion into this site was observed in an initial screening of single cell clones The efciency of HE-induced targeted gene insertion in other sites is presently under evaluation 91 Cell Synchronization by Serum Shock Facilitates rAAV-Mediated Gene Targeting Yonglun Luo,1,3 Emil Kofod-Olsen,2 Jenny Blechingberg,1 Rikke Christensen,1 Nicklas Heine Staunstrup,1 Lars Bolund,1,3 Charlotte Brandt Sørensen.1 Department of Human Genetics, Aarhus University, Aarhus C, Denmark; 2Department of Medical Microbiology and Immunology, Aarhus University, Aarhus C, Denmark; 3Hua Da/BGI, Shenzhen, China Recombinant adeno-associated virus (rAAV) mediated gene targeting greatly facilitates homologous recombination (HR) compared to plasmid DNA methods However, it is still hampered by low targeting and high random integration frequency It has been S35 ... inserted into the same location Interestingly, the lacZ target with the “replacement” inactivating mutation supported nick-induced HR at a 10-fold higher rate than the target with the “insertion”... results suggest that is incorrect Both the observation that nicks can stimulate HR with lower toxicity than DSBs and the observation that target site design effects nick-induced HR but not DSB-induced... contrast, the toxicity of the nicking endonuclease was low and was not distinguishable from the toxicity of an inactive endonuclease or the toxicity of an empty lentivirus vector expressing only the mCherry