93. Chromatin Structure of Two Genomic Sites for Targeted Transgene Integration in Induced Pluripotent Stem Cells

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 efciency should be promoted. To test this hypothesis, we rst evaluated the synchronizing effect of serum starvation (SST) (0.5% FCS for 2 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targeting exon 11 in porcine broblasts used as nuclear donors in Somatic Cell Nuclear Transfer cloning (Y. L et al., unpublished results).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 specic 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 2 days until 2 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 specic targeting efciency. The frequency of BRCA1 targeting for the control was 32.5% (50 BRCA1 KO clones/154 G418r clones screened).The targeting efciency 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 benecial 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.

1

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 prole of PhiC31 integrase in mammalian cells inducible systems were established, the N-terminal catalytic domain was mutated, DNA shufing 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 efciency 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 5 mutants with up to 3-fold enhanced excision activity. Importantly, we also identied mutants showing up to 2.5-fold recombination activities between attB and 3 previously described attP’ sites on chromosomal positions 19q13.31, 2q11.2, and 12q22 in the mammalian genome, indicating that there may be enhanced specicity for these hot spots. With respect to integration into mammalian genomes initial screens identied mutants displaying up to 1.7-fold increased somatic integration efciencies in HeLa cells. However, combination of benecial mutations in addition to optimization of the integrase plasmid dose further enhanced integration efciencies up to 6-fold. Notably, integration efciencies were cell line dependent because the tested mutants showed varying integration efciencies in human kidney, colon, and liver derived cell lines. We also identied 3 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 benecial 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 benecial 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 efcacy may be species dependent. In summary, we identied 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 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 5 (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 congurations 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 conguration, the immune cell-restricted CCR5 gene, has a predominantly inactive chromatin conguration in iPSCs and Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy

TARGETING GENE MODIFICATION AND INTEGRATION cord-blood derived hematopoietic stem cells. This suggests that the CCR5- site is relatively unsuited for therapeutic transgene insertion using ZFNs. Furthermore, we used mChIP assays to characterize cellular factors that are potentially involved in Rep78-mediated gene targeting in iPSCs and HSCs. First data demonstrate that the Repbinding site within AAVS1 displays high occupancy of polymerase II, indicating a potential link between Rep-mediated replication/ integration and pol-II transcription.

94. Targeted Plasmid Integration into the PiggyBac Transposon by an Engineered Zinc Finger Recombinase

Charles A. Gersbach,1,2 Russell M. Gordley,1 Thom Gaj,1 Andrew C. Mercer,1 Carlos F. Barbas, III.1 1 Departments of Chemistry & Molecular Biology, The Scripps Research Institute, La Jolla, CA; 2Department of Biomedical Engineering, Duke University, Durham, NC. Precise modification of mammalian genomes is central to numerous applications in medicine, biotechnology, and basic research. We have engineered novel synthetic enzymes for this purpose, consisting of a recombinase catalytic domain fused to a programmable zinc nger DNA-binding protein. We have recently demonstrated that these zinc nger recombinases (ZFRs) are capable of directing plasmid integration into a targeted genomic location with unprecedented specicity. In contrast to zinc nger nucleases, the ZFRs autonomously catalyze the integration reaction and do not require the induction of DNA damage or endogenous DNA repair pathways. In this study, the piggyBac transposase system was used in combination with ZFRs to integrate reporter plasmid into specic locations in the genomes of several human and mouse cell lines. Both the specicity and efciency of plasmid integration were assessed by reporter gene expression, direct analysis of genomic DNA, and derivation of clonal integration events. The overwhelming majority of plasmid integration events occurred at the ZFR target site. Targeted integration was also highly efcient relative to background levels of non-specic plasmid integration. Mapping the genomic location of integration events demonstrated the diversity of successful ZFR target sites across the human genome. Given the variety of recent successful uses of transposase systems, we expect there to be several promising applications of this approach. The development of this site-directed and nonviral gene delivery system is signicant for establishing enhanced methods for gene therapies, basic genetic studies, and cell line engineering for biopharmaceutical production.

95. Development of Autonomously Working Zinc-Finger Nuclease Pairs for Targeted Genome Engineering

Cem Söllü,1 Kaweh Montazami-Astaneh,1 Morgan L. Maeder,2 Stacey Thibodeau-Beganny,2 J. Keith Joung,2 Toni Cathomen.1 1 Dept of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2Dept of Pathology, Massachusetts General Hospital, Charlestown, MA. Zinc-nger nucleases (ZFNs) have proven useful for therapeutic genome engineering in a variety of cell types, including human stem cells. ZFNs consist of a non-specic FokI nuclease domain and an engineered zinc-nger DNA-binding domain. As the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The use of ZFNs can be associated with signicant cytotoxicity, partially attributed to unspecic DNA cleavage by ZFN homodimers. To expand the application range of ZFNs, we aimed at developing autonomously working ZFN pairs that will allow us to direct two ZFN pairs simultaneously to two different target sites without any cross-reactivity. We have used a panel of in silico, in vitro, and in vivo assays to characterize the consequence Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy

of structure-based changes in the architecture of the FokI cleavage domain with respect to ZFN activity and ZFN-associated toxicity, to identify ZFN pairs that combine high cleavage activity with low toxicity. In silico energy calculations revealed that point mutations in the FokI dimer interface of all four published ZFN variants weaken dimerization and prevent the formation of homodimers. In vitro cleavage assays and cellular gene targeting assays conrmed that homodimerization was prevented for all variants but pointed out that the activity of one published variant was considerably attenuated. Importantly, we were able to identify two variant ZFN pairs that showed no cross-reactivity between each other. Initial experiments with such ZFN variants designed to recognize the endogenous human HoxB13 locus demonstrated that it is possible to induce targeted chromosomal deletions. These results demonstrate the utility of autonomous ZFN variants in rational genome engineering.

96. Adeno-Associated Virus-2 (AVV2) Integrase Mediates Specic Integration to Locus AAVS1 on Chromosome 19q13.3 in a Hematopoietic Cell Line Ashley E. Dunfee, Joshua R. Clevenger, Cynthia E. Dunbar, Andre Larochelle. NHLBI, Hematology Branch, National Institutes of Health, Bethesda, MD.

Gene therapy has already demonstrated considerable efcacy in the treatment of hematopoietic disorders such as X-SCID, ADASCID and CGD. However, the retroviral vectors utilized in these therapies can integrate into the host genome in a semi-random fashion, which can result in dysregulated expression of nearby genes, including many which may lead to cancer. In an effort to avoid insertional mutagenesis, we investigated the use of a selected integrase, the AAV2 integrase, to effect targeted integration in the genome of human hematopoietic cells. This integrase is known to result in site-specic integration in the genomic locus AAVS1 located on chromosome 19q13.3, a region of the genome not associated with cancer formation, providing a potentially safer approach for gene therapy of hematopoietic disorders. To test this approach, we transfected the human hematopoietic cell line K562 by nucleofection with two plasmids: one plasmid containing the GFP reporter gene anked by the AAV2 ITRs; and one plasmid containing the AAV2 integrase gene (AAV2 integrase group). Both the AAV2 integrase and GFP genes were under the control of the EF1α promoter. Two control lines were also established by transfecting the same GFP plasmid in combination with a defunct AAV2 integrase plasmid (negative control group) or with a plasmid expressing an integrase derived from the bacteriophage PhiC31 with no known specicity to chromosome 19q13.3 (PhiC31 integrase group). One day after transfection, 40% of K562 cells expressed GFP in both the AAV2 integrase and negative control groups, and 75% GFP+ cells were detected in the PhiC31 integrase group. Two months later, 2% and 1% of transfected cells maintained detectable GFP expression in the AAV2 integrase group and PhiC31 integrase group, respectively, while only 0.1% of the negative control group expressed GFP. GFP+ cells were subsequently sorted via FACS, cultured for up to 6 months and assessed by ow cytometry for GFP positivity at regular intervals. In the AAV2 integrase group, more than 98% of the sorted cells remained GFP+ after 6 months in culture compared to 40% in the negative control group. Sorted cells were assayed by PCR for detection of integration in locus AAVS1 using a forward primer specic to the integrated GFP gene and a reverse primer specic to various regions of the AAVS1 locus. A PCR product was detected only in the AAV2 integrase group, conrming specicity of integration at this locus. Additionally, sorted cells were assayed by FISH using dual hybridization with probes targeting GFP and chromosome 19 to detect integration to chromosome 19q. In 8 of 10 (80%) metaphases analyzed, integration was detected on chromosome 19q in cells from S37