91 Cell Synchronization by Serum Shock Facilitates rAAV Mediated Gene Targeting Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S34 TARGETIN[.]
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 specic 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 specicities that can be autoreactive Thus, we developed ZFNs specic 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 efciently achieved in CD3neg sorted cells (>45%) and resulted in higher expression of the tumor specic 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-specic 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 efcient 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 efcient 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 specic 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 proling Three near-cognate sites were identied that cleaved with high efciency 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 decient 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 efciency 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 efciency 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 TARGETING GENE MODIFICATION AND INTEGRATION established that HR is cell cycle dependent, in which HR mainly occurs during the late S/G2 phase while non-homologous end joining (NHEJ) plays a predominant role in the G1/early S phase Thus, we hypothesize that if the cell cycle could be synchronized and the rAAV mediated transduction was performed in a period when cells transit form G0/G1 into late S/G2, gene targeting efciency should be promoted To test this hypothesis, we rst evaluated the synchronizing effect of serum starvation (SST) (0.5% FCS for days) followed by serum shock (SSH) (50% FCS for 2h) on primary porcine broblasts The portion of G0/G1 and G2/M cells after SST was approx 88.4% and 9.0%, changing to 39.7% and 52.5% at 22h after SSH Based on this result, we performed time-course transductions using an rAAV/BRCA1 KO viral vector with G418 as the selective marker This rAAV/BRCA1 KO construct has been generated by us with the objective of producing BRCA1 KO pigs by targeting exon 11 in porcine broblasts used as nuclear donors in Somatic Cell Nuclear Transfer cloning (Y L et al., unpublished results) We have observed that the specic BRCA1 targeting frequency with this construct in a normal transduction procedure was approx 35% (i.e 35% of the G418 resistant (G418r) clones showed HR) To evaluate the effects of SST-SSH on gene targeting, 3×105 cells were subjected to SST followed by SSH Cells were transduced with 1×1011 viral vector particles before SST (control) and at 0h, 2h, 6h, 10h, or 22h after SSH 24 hrs after transduction, cells were trypsinized and split into gelatin coated 96-well plates (n = 9) Cells were selected with G418 from days until weeks after splitting The BRCA1 targeting frequency was determined by PCR Based on this investigation, we found that the development of G418r clones (number of G418r clones per 96-well plate, n = 9) was dependent on time after SSH The frequencies for control, 0h, 2h, 6h, 10h and 22h were 17.1 %, 7.6%, 8.2%, 14.8%, 18.7% and 28.6%, respectively PCR screening of the G418 resistant clones further showed that SSH induced a time-dependent increase in BRCA1 specic targeting efciency The frequency of BRCA1 targeting for the control was 32.5% (50 BRCA1 KO clones/154 G418r clones screened).The targeting efciency at 0, 2, 6, 10 and 22 hrs after SSH were determined in two independent experiments The results were: 33.3% (2/6) and 49.3% (34/69) at 0h, 57.14% (8/14) and 47.3% (35/74) at 2h, 76.47% (13/17) and 53.5% (68/127) at 6h, 45.45% (10/22) and 39.7% (52/131) at 10h, 46.67% (14/30) and 47.3% (71/150) at 22h In conclusion, we have shown that cell synchronization by serum shock is capable of enhancing the rAAV mediated gene targeting frequency This approach might be benecial for the application of rAAV mediated gene therapy by HR 92 Critical Amino Acid Residues within the PhiC31 Integrase DNA Binding Domain Affect Recombination Activities in Mammalian Cells Raphael Liesner,1 Wenli Zhang,1 Nadja Noske,1 Anja Ehrhardt.1 Virology, Max von Pettenkofer-Institute, Munich, Germany The bacteriophage-derived PhiC31 integrase system represents an attractive tool for site-directed recombination in mammalian cells Its recombination process is based on recombination between the attachment site attB within an episomal substrate plasmid and either the bacteriophage-derived wild type attachment site attP or pseudo attP attachment sites (attP’) present in the mammalian genome To improve potency and the toxicity prole of PhiC31 integrase in mammalian cells inducible systems were established, the N-terminal catalytic domain was mutated, DNA shufing approaches were performed, codon-optimized version were generated, and a NLS signal was fused to the PhiC31 protein In the present study we aimed at increasing safety and efciency of PhiC31 integrase-mediated recombination by mutating for the rst time the DNA binding domain at the C-terminus Utilizing an alanine mutagenesis approach, we generated 22 PhiC31 point mutants which were screened for activities in mammalian cells S36 Intramolecular excision assays based on recombination between attB and wild type attP revealed mutants with up to 3-fold enhanced excision activity Importantly, we also identied mutants showing up to 2.5-fold recombination activities between attB and previously described attP’ sites on chromosomal positions 19q13.31, 2q11.2, and 12q22 in the mammalian genome, indicating that there may be enhanced specicity for these hot spots With respect to integration into mammalian genomes initial screens identied mutants displaying up to 1.7-fold increased somatic integration efciencies in HeLa cells However, combination of benecial mutations in addition to optimization of the integrase plasmid dose further enhanced integration efciencies up to 6-fold Notably, integration efciencies were cell line dependent because the tested mutants showed varying integration efciencies in human kidney, colon, and liver derived cell lines We also identied PhiC31 mutants which were recombination defective in all applied assays, suggesting that these amino acid residues are essential for functionality of PhiC31 integrase in mammalian cells As a further step most benecial mutants were tested in vivo in mouse liver utilizing hydrodynamic tail vein injection of a human coagulation factor IX (hFIX) encoding substrate plasmid and the respective PhiC31 variants Stability of transgene expression was monitored and rapid cell cycling in murine liver was induced by carbon tetrachloride injection However, the two tested PhiC31 mutants showed no benecial effect with respect to persistence of transgene expression This suggests that mutants optimized for somatic integration in human cells may not be advantageous for applications in murine cells and that PhiC31 efcacy may be species dependent In summary, we identied critical amino acid residues within the PhiC31 DNA binding domain With respect to site-directed recombination and genome engineering these ndings have important implications for rational and improved PhiC31 protein design 93 Chromatin Structure of Two Genomic Sites for Targeted Transgene Integration in Induced Pluripotent Stem Cells Ruan van Rensburg,1 Oleg Denisenko,1 Karol Bomsztyk,1 Andre Lieber.1 Medicine, University of Washington, Seattle, WA We are pursuing two adenovirus vector-based approaches to achieve targeted integration of transgenes controlled by the b-globin locus control region (LCR) The rst approach utilizes that ability of the AAV protein Rep78 to mediate targeted gene integration in the absence of adverse insertional mutagenesis or functional consequences Rep78 catalyzes DNA nicking and synthesis at the AAVS1 site, located within the ubiquitously expressed mysosinbinding subunit 85 (MBS85) gene The second approach is based on a zinc-nger nuclease (ZFN) that mediates double strand breaks within the Chemokine (C-C motif) receptor (CCR5) gene on chromosome 3, which is expressed predominantly in T-cells, macrophages and dendritic cells Notably, the genetic inactivation of the CCR5 gene has no pathological side effects For both approaches to be successful the corresponding genomic target sites must be transcriptionally active areas for the integrants of Rep78 and CCR5-ZFN, respectively In this study we therefore examined the chromatin congurations of the AAVS1 and CCR5-ZFN sites in a range of human cell lines, including induced pluripotent stem cells (iPSCs) and hematopoietic stem cells (HSCs) from different donors Matrix chromatin immunoprecipitation (mChIP) assays were performed with various histone markers, including histone H3, H3 tri methyl K9 (H3K9m3), H3K9/14 acetylated, and H3 tri methyl K27 (H3K27m) The analyses clearly indicate that while the AAVS1 site possesses an active chromosome conguration, the immune cell-restricted CCR5 gene, has a predominantly inactive chromatin conguration in iPSCs and Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... that cell synchronization by serum shock is capable of enhancing the rAAV mediated gene targeting frequency This approach might be benecial for the application of rAAV mediated gene therapy by. .. transductions using an rAAV/ BRCA1 KO viral vector with G418 as the selective marker This rAAV/ BRCA1 KO construct has been generated by us with the objective of producing BRCA1 KO pigs by targeting exon... hypothesize that if the cell cycle could be synchronized and the rAAV mediated transduction was performed in a period when cells transit form G0/G1 into late S/G2, gene targeting efciency should