415 Further Improvements and Applications of OPEN (Oligomerized Pool ENgineering) A Rapid, Robust, and Publicly Available Method for Engineering Customized Zinc Finger Nucleases Molecular Therapy Volu[.]
GENE REGULATION regions from gene expression profiles in experimental arthritis and experimentally verified a panel of ten identified promoters We first performed gene expression profiling of synovial knee joint tissues from mice with collagen-induced arthritis to identify the genes that show at least tenfold regulation during disease progression We then used k-means clustering to group these genes into six distinct expression profiles Next, we introduced a filtering based on the presence of a putative TATA-box between positions -32/-29 in the upstream promoter regions of the murine genes and their human orthologs The over-represented cis-regulatory elements in the proximal promoters (-500/+200) that putatively govern the distinct expression profiles were identified using an algorithm that takes advantage of spatial and phylogenetic conservation of sequences Based on this algorithm, we constructed lentiviral luciferase reporters containing proximal promoter regions that were predicted to be predominantly regulated by the transcription factors NFκB (Cxcl1, Cxcl5, Il1b), AP-1 (Mmp3, Mmp13, Timp1, Tnfaip6) or C/EBPβ (Saa3, Chi3l1, Has1) Nine out of ten promoters were responsive to a pro-inflammatory stimulus in murine fibroblasts and macrophages The promoter regions of Saa3, Cxcl1 and Mmp3 showed the strongest, more than tenfold, increase over basal promoter activities For in vivo validation, lentiviral reporters were injected in knee and ankle joints of C57Bl/6 mice and the kinetics of luciferase expression during acute inflammation were determined by optical imaging or ex vivo luciferase assays The promoter activities were rapidly and strongly induced during onset of inflammation and significantly decreased when inflammation waned Of great value for human gene therapy, the relative Saa3 promoter response was significantly increased in primary RA synovial fibroblasts derived from patients with a high synovial inflammatory gene expression profile This study highlights the value of a bioinformatics approach in design of transcriptionally targeted gene therapy for disease We expect the novel vectors to be widely applicable in both basic and translation research in human and experimental arthritis 414 PARP-1 Contributes to the Barrier Function of a Vertebrate Chromatin Insulator David W Emery, Karol Bomsztyk, Mari Aker Divisions of Medical Genetics and UW Medicine Lake Union, Department of Medicine, University of Washington, Seattle, WA The prototypic chromatin insulator cHS4 has proven effective at reducing silencing chromosomal position effects in a variety of settings Most of this barrier insulator activity has been mapped to a 250 bp core region, as well as to several proteins that bind this region However, recent studies from our laboratory found that an extended 400 bp core region of the cHS4 element was necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors [Aker et al., Hum Gene Ther 18:333, 2007] In more recent studies, electrophoretic gel mobility shift assays reveled specific DNA protein binding activities associated with the distal portion of this extended core region Affinity purification and tandem mass spectrometry studies lead to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1), an abundant nuclear protein that has the capacity to bind DNA through zinc finger motifs, and to catalyze the addition of poly(ADP)-ribose chains to itself and other proteins The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro, and by chromatin immunoprecipitation studies in vivo Footprinting studies using ssDNA probes suggest that PARP-1 binding to this extended cHS4 core region is specific to a unique DNA cruciform secondary structure, rather than a primary sequence Functional studies with gammaretroviral reporter vectors in cell lines showed that cHS4 barrier activity was abrogated upon deletion or mutation of the putative PARP-1 binding site, reducing the frequency of cells S162 expressing vector GFP 2-fold to the levels seen without the insulator Transduction studies in primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was also abrogated following treatment with a PARP inhibitor, reducing the level of vector GFP expression to 7-fold to an average of 8-45 mean fluorescent units, levels seen with the uninsulated control Finally, barrier activity of the cHS4 element was also found to be abrogated in similar progenitor transduction studies using bone marrow from Parp1-null mice In this case, the frequency of cells expressing vector GFP from the cHS4-insulated vector decreased from an average of 51-59% in cells from normal mice to an average of 30-39% in cells from Parp1 null mice (compared to 23-30% for the uninsulated control) All of these differences were statistically significant PARP-1 has been shown by others to play significant roles in chromatin remodeling and transcriptional control, with some evidence suggesting it may play a role in enhancer-blocking insulator activity Taken together, our studies demonstrate that binding of PARP-1 also plays a key functional role in the barrier activity of the prototypic cHS4 chromatin insulator, and helps to explain why the extended 400 bp cHS4 core exhibits more activity than smaller versions of this element 415 Further Improvements and Applications of OPEN (Oligomerized Pool ENgineering): A Rapid, Robust, and Publicly Available Method for Engineering Customized Zinc Finger Nucleases Morgan L Maeder,1 Jing-Ruey J Yeh,2 Jonathan E Foley,1 Jizhong Zou,3 Stacey Thibodeau-Beganny,1 Linzhao Cheng,3 Randall T Peterson,2 J Keith Joung.1 Molecular Pathology Unit, Massachusetts General Hospital/ Harvard Medical School, Charlestown, MA; 2Cardiovascular Research Center, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA; 3Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD Engineered zinc finger nucleases (ZFNs) can induce highly efficient genome modifications in a wide variety of cell types ZFNs consist of a customized DNA-binding zinc finger array fused to a non-specific nuclease domain ZFN-induced double-stranded DNA breaks can be repaired by either non-homologous end-joining or homologous recombination, processes that can be used to introduce desired alterations with very high efficiencies at or near the site of the break The development of a rapid, robust, and publicly available capability to re-engineer the DNA-binding specificities of ZFNs is a critically important requirement for the practice of this technology We have recently described OPEN (Oligomerized Pool ENgineering), a userfriendly selection-based method for engineering ZFNs that accounts for the context-dependent behavior of individual zinc fingers (Maeder et al., 2008) ZFNs engineered using OPEN have been used to induce highly efficient modification of numerous endogenous human, plant, and zebrafish genes (Maeder et al., 2008; Townsend et al., 2009; Foley & Yeh et al., 2009) Here we describe recent alterations to the OPEN protocol which have allowed us to increase the speed of the method and to improve the scalability of selections These modifications, which permit the use of multi-well plates/blocks and multi-channel pipets, now enable us to perform selections for 48 target sites in less than eight weeks time We have used this higher-throughput method to engineer ZFNs for target sites in nine endogenous zebrafish genes To date, we have shown that many of these OPEN ZFNs can efficiently modify their endogenous gene targets in somatic cells and that these alterations can be passed through the germline In addition, we report on the engineering of new ZFNs targeted to the endogenous human PIG-A gene Nonsense mutations in PIG-A are associated with paroxysmal nocturnal hemoglobinuria (PNH) disease We have shown that OPEN ZFNs targeted to PIG-A can stimulate gene targeting in both human somatic and stem cells Taken together, our results enable Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy OLIGONUCLEOTIDE THERAPIES broader and more rapid use of the OPEN ZFN selection methodology and provide additional support for the potential use of ZFNs as an important approach for gene therapy 416 Treatment of Canine Leukocyte Adhesion Deficiency Using a SIN Lentiviral Vector and Human CD18 Promoter Expressing Canine CD18 Michael J Hunter,1 Laura M Tuschong,1 Cedar J Fowler,2 Everette J R Nelson,1 Tanya H Burkholder,3 Thomas R Bauer, Jr.,1 Dennis D Hickstein.1 Experimental Transplantation and Immunology, National Cancer Institute, Bethesda, MD; 2HHMI/NIH Research Scholars Program, Howard Hughes Medical Institute, Chevy Chase, MD; Department of Veterinary Resources, National Institutes of Health, Bethesda, MD Hematopoietic stem cell gene therapy would be enhanced by the development of vectors harboring tissue-and developmental stagespecific cellular promoters to express the therapeutic transgene in the target cell population This approach would mitigate the potential adverse effects of inappropriate tissue expression and might be expected to reduce insertional activation of nearby oncogenes that lead to oligoclonal hematopoiesis and leukemia To develop and test a modified promoter cassette with the features described above for our target disease canine leukocyte adhesion deficiency (CLAD), we cloned portions of the human CD18 promoter (1 Kb, 776 bp, and 306 bp) into a SIN lentiviral vector upstream of the canine CD18 cDNA, and used this vector to transduce CLAD CD34+ cells In CLAD, defects in the leukocyte integrin CD18 result in the inability to express CD11/CD18 heterodimers on the leukocyte surface leading to lifethreatening bacterial infections Transduction of CLAD CD34+ cells in vitro with the SIN lentiviral vector with the kb human CD18 promoter resulted in the highest percentage of CD18+ cells; nearly 15% of the CLAD CD34+ cells were CD18+ when assessed days after an overnight transduction We used the SIN lenti vector with the Kb human CD18 promoter to treat two dogs with CLAD Autologous, CLAD CD34+ cells were transduced overnight and infused following a single, non-myeloablative dose of 200cGy total body irradiation (TBI) The percentage of CD18+ leukocyte compartments weeks following infusion of the two dogs were comparable: dog A1, CD18+/ PMN 0.3%, CD18+/CD3+ cells 0.4%, CD18+/B-cells 0.7%, CD18+/ CD14+ cells 0.9%; dog A2, CD18+/PMN 0.5%, CD18+/CD3+ cells 0.8%, CD18+/B-cells 1.2%, CD18+/CD14+ cells 1.4% Both treated dogs are now months of age and have had correction of the CLAD phenotype In contrast, untreated CLAD dogs succumb to overwhelming infection within a few months of life Reversal of the CLAD phenotype with low numbers of CD18+ neutrophils results from selective migration of CD18+ neutrophils from the blood into the tissues Although both CLAD dogs in this study have had a clinical response, additional regulatory elements of the human CD18 promoter/enhancer will be required to ensure long-term reversal of the phenotype in this and other genetic leukocyte diseases These studies represent requisite translational studies in the development of new vector designs for the treatment of children with the human counterpart of CLAD, namely LAD 417 Efficient MGMTP140K-Mediated Selection of Long Term Repopulating Cells in a Nonhuman Primate Model Brian C Beard,1 Grant D Trobridge,1,2 Megan L Welsh,1 HansPeter Kiem.1,2 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Department of Medicine, University of Washington, Seattle, WA In vivo selection of genetically modified hematopoietic repopulating cells has many potential therapeutic applications For some applications which require relatively high levels of gene marking, such as hemoglobinopathies, in vivo selection may be required to increase initially low levels of gene-modified cells Here we demonstrate efficient post-transplantation selection of long-term hematopoietic repopulating cells using methylguanine methyltransferase (MGMTP140K) in a primate model In vivo selection was studied in macaques (M nemestrina) that received CD34-enriched cells transduced with VSVG-pseudotyped HIVderived lentivirus vectors expressing MGMTP140K and a reporter gene green fluorescent protein (GFP) or MGMTP140K only Two macaques were conditioned with a myeloablative dose of total body irradiation and a third macaque was conditioned with a nonmyeloablative dose of busulfan (4 mg/kg/day for days) After stable engraftment monkeys were treated with O6-benzylguanine (O6BG) and BCNU Following myeloablative transplantation the monkeys transplanted with cells gene-modified with a vector expressing MGMTP140K and GFP, in vivo selection was determined by flow cytometry In one monkey (following cycles of O6BG/ BCNU) the granulocytes rose from ∼20% to 70% and in the lymphocytes from ∼20% to 50% In the second monkey (following a single cycle of O6BG/BCNU) the granulocytes rose from ∼25% to 42% and in the lymphocytes from ∼12% to 22% Following nonmyeloablative transplantation and a single cycle of O6BG/BCNU the monkey transplanted with cells gene-modified with the vector expressing only MGMTP140K increased overall gene marking, determined by real time (RT)-PCR, in total white blood cells rose from a provirus copy number of 0.04 (∼4% gene marking) to 0.16 (∼16% gene marking) Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed Importantly, multilineage selection of hematopoietic cells was achieved and clonality studies are underway using a combination of LAM-PCR and a modified whole genome pyrosequencing approach In summary, MGMT selection is efficient and well tolerated in macaques and these large animal studies should be highly predictive for clinical applications and will help to further improve HSC gene therapy Oligonucleotide Therapies 418 Application of Therapeutic Artificial miRNAs in the CNS: Non-Allele-Specific Silencing of Mutant and Wildtype Huntingtin Demonstrates Therapeutic Efficacy in Huntington’s Disease Mice Ryan L Boudreau,1 Jodi L McBride,1 Ines Martins,1 Shihao Shen,1 Yi Xing,1 Barrie J Carter,2 Beverly L Davidson.1 University of Iowa, Iowa City, IA; 2Targeted Genetics, Seattle, WA RNA interference (RNAi) provides a promising therapeutic approach to treat several human diseases However, the safety of RNAi-based therapies remains a concern Previously, we compared the efficacy and safety of short-hairpin RNA (shRNA) and artificial microRNA (miRNA) expression vectors in vitro and in vivo We found that shRNAs are more potent but induce toxicity in cell cultures and in mouse brain, whereas artificial miRNAs are expressed at lower levels and display better safety profiles We have since tested the Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S163 ... Two macaques were conditioned with a myeloablative dose of total body irradiation and a third macaque was conditioned with a nonmyeloablative dose of busulfan (4 mg/kg/day for days) After stable... THERAPIES broader and more rapid use of the OPEN ZFN selection methodology and provide additional support for the potential use of ZFNs as an important approach for gene therapy 416 Treatment of. .. efficacy and safety of short-hairpin RNA (shRNA) and artificial microRNA (miRNA) expression vectors in vitro and in vivo We found that shRNAs are more potent but induce toxicity in cell cultures and