GENE REGULATION: GENE TARGETING AND RNA 374 Knockdown of Shp-2 Expression by siRNA in Mouse Embryonic Stem Cell Derived EB Cells Affects Their Hematopoietic Development Gang-Ming Zou,1 Rebecca J Chan,1 W Christopher Shelley,1 Mervin C Yoder.1 Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN Shp-2 is a member of a small subfamily of cytoplasmic Src homology (SH2) domain-containing protein tyrosine phosphatases To address the question of whether Shp-2 is required for normal hemangioblast, primitive, and definitive hematopoietic development, we used Shp-2 siRNA to knockdown Shp-2 gene expression in embryonic stem (ES) derived embryoid body (EB) cells We found that a pool of (but not single siRNA) Shp-2 siRNA at a concentration of 50 nM/ml was efficient to knockdown Shp-2 expression in EB cells The density of Shp-2 protein in western blot assay was decreased about 10 fold in Shp-2 siRNA treated cells compared with cells treated with a scrambled control Shp-2 siRNA The frequency of growth factor-dependent hemangioblast formation were decreased fold following cell treatment with Shp-2 siRNA (112.6 ± 11.7 vs 31 ± 1), but not by scrambled Shp-2 siRNA (112.6 ± 11.7 vs 114 ± 12) Basic fibroblast growth factor (bFGF) promotes hemangioblast development As Shp-2 is involved in the upstream signaling of bFGF receptor activation, we then asked whether Shp2 was involved in this bFGF mediated hemangioblast effect Addition of bFGF at doses of 10 and 20 ng/ml significantly increased (121.6 ± 4.7 vs 232.3 ± 10) and (121.6 ± 4.7 vs 281.3 ± 4.1) (compared with control siRNA treated cells) the frequency of hemangioblast formation However, knockdown of Shp-2 gene expression by siRNA blocked the bFGF effect on hemangioblast development In primitive hematopoietic assays, knockdown of Shp-2 expression in day-6 EB cells resulted in a ( 350 ± 12.5 vs 173 ± 16.3) fold decrease in EPOdependent Ery-P colonies, a (25 ± 2.3 vs 7.2 ± 1.8) fold decrease in Ery-D colonies and a ( 23 ± 1.8 vs 4.1 ± 0.6) fold decrease in GM-CSF-dependent CFU-GM colonies Altogether, these data demonstrate that Shp-2 is required in hemangioblast, primitive, and definitive progenitor hematopoietic development Shp-2 is also involved in bFGF regulation of hemangioblast development We conclude that Shp-2 plays a critical role in hemangioblast, primitive, and definitive hematopoiesis 375 Targeted Gene Repair Induces the DNA Damage Response Pathways through the Activation of ATM, and the Stalling of Replication Forks Leading to a Blockage of Inherited Correction Luciana Ferrara,1 Julia Engstrom,1 Erin Brachman,1 Miya Drury,1 Eric B Kmiec.1 Biological Sciences, University of Delaware, Newark, DE We have been investigating the mechanism of targeted gene repair in mammalian cells using an eGFP reporter system The protocol involves the electroporation of single-stranded oligonucleotides into an adenocarcinoma cell line containing integrated copies of a mutant eGFP gene Correction of the point mutation is directed by the oligonucleotide in a reaction involving members of the homologous recombination (HR) pathway The electroporation of single-stranded vector and the presence of free ends of DNA induces HR as the cell perceives that DNA damage has occurred This response sets in motion a cascade of events including the activation of ATM and the cessation of DNA replication Surprisingly, we find that cells bearing a corrected allele, verified at the phenotypic and genotypic levels are preferentially stalled in S phase and at the S/G2 border Uncorrected cells resume active replication and mitosis, which essentially leads to a dilution of corrected cells as a S146 function of time Thus, perceptions that the gene repair has failed in certain systems can be accounted for by such a dilution phenomenon These results also help explain the variable levels of correction seen during the early days of gene repair Most importantly, these observations clearly have a long-term and profound effect on the potential for using synthetic oligonucleotides in ex vivo gene therapy applications 376 Various Aspects of the Single-Stranded DNA Fragment-Mediated Gene Correction Hiroyuki Tsuchiya,1,2 Hideyoshi Harashima,1,2 Hiroyuki Kamiya.1,2 Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan; 2CREST, Japan Science and Technology, Japan The gene correction strategy, by which mutations are converted to the normal sequences in living cells, is expected as an important technology for gene therapy and biotechnology We have developed a new gene correction method by using a sense single-stranded (ss) DNA fragment (fSense) prepared from phagemid DNA (Tsuchiya H, et al J Gene Med., in press) This ss DNA fragment provided 12-fold higher gene correction efficiency as compared with a doublestranded (ds) PCR fragment used in the conventional small-fragment homologous replacement method However, at the present time, very limited information is available about factors affecting the correction efficiency and mechanisms involved in the ss DNA fragment-mediated gene correction In the present study, we performed several experiments to reveal the factors and mechanisms how the ss DNA fragments correct mutations of target genes Moreover, we examined the potency of new DNA fragments modified based on the outcomes of the mechanistic studies First, we investigated the effects of the transcription of the target gene, of the DNA modifications (5’-phosphate and N6-methyladenine [mA]) of fSense and of the length of ss DNA fragments on the gene correction Second, we examined whether fSense was integrated into the homologous strand of the target gene It was revealed that the transcription did not affect the gene correction while the mA, but not 5’-phosphate, significantly stimulated the gene correction (2fold increase), and that there was an optimum value in the length (400–1200 nt) Additionally, fSense was indeed integrated into the target gene, indicating that a homologous recombination (HR) pathway was, at least in part, involved in the gene correction process This finding led us to further modifications of the ss DNA fragments based on the HR mechanism Currently, the newly modified DNA fragments are under investigation These results suggest that further improvement in the gene correction efficiency can be achieved by modification(s) of DNA fragments based on the correction mechanism, the HR pathway 377 Developing siRNAs to the E2Fs to Control Vascular Growth and Remodeling Paloma H Giangrande,1,2 JianXin Zhang,1 Juliana Layzer,1 Otto Hagen,1 Joseph R Nevins,2 Bruce A Sullenger.1 Surgery, Duke University DUMC, Durham, NC; 2Molecular Genetics and Microbiology, Duke University DUMC, Durham, NC Ischemia represents a major causative condition in cardiovascular and cerebrovascular syndromes, which triggers proliferative and degenerative alterations of the vascular wall leading to the stenosis of arteries supplying blood to vital organs Surgery is often the final treatment modality to restore blood supply to the ischemic organ via by-passing the site of major stenosis using arterial or venous grafts Alternatively, percutaneous transluminal catheters can be used to restore blood flow to an organ by reopening the stenosis Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy GENE REGULATION: GENE TARGETING AND RNA from the inside of the arterial lumen using the methods of balloon angioplasty alone or in combination with stent implantation However, the major limitation of these procedures is that they themselves trigger accumulation and proliferation of vascular smooth muscle (VSM) cells from the tunica media to tunica intima of the vascular wall leading to restenosis or vein-graft failure in a significant percentage of all cases This pathological process is known as intimal hyperplasia Limiting intimal hyperplasia in grafted vessels or vessels following angioplasty is a critically important therapeutic target A number of studies have shown that growth suppression action of the retinoblastoma tumor suppressor protein (Rb) and other Rb family members is dependent on their ability to regulate the E2F family of transcription factors (E2F1-7) Recently it has been demonstrated that inhibition of the entire family of E2F proteins can reduce intimal hyperplasia Here we show that inhibition of individual (or subsets of the) E2Fs can enhance or reduce intimal hyperplasia following vessel damage or grafting Specifically, we show that siRNAs to E2F1 and E2F3 act as specific inhibitors of vascular smooth muscle cell proliferation in vitro and in vivo Importantly, these studies have lead to a better understanding of how distinct E2F activities promote or repress vascular smooth muscle cell proliferation during intimal hyperplasia and have yielded lead siRNAs that establish a therapeutic proof of principal in animals undergoing by-pass surgery or angioplasty In conclusion, this work has facilitated the development of more potent and more specific inhibitors of this pathological process 378 Retargeting Mobile Group II Introns to Repair Mutant Genes Monique N Kierlin,1 John P Jones III,1 Rob G Coon,2 Jiri Perutka,2 Alan M Lambowitz,2 Bruce A Sullenger.1 Departments of Surgery and Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC; Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas, Austin, TX Retroposable elements such as retroviral and lentiviral vectors have been employed for many gene therapy applications Unfortunately, such gene transfer vectors integrate genes into many different DNA sequences and unintended integration of the vector near a growth promoting gene can engender pathological consequences For example, retroviral vector-mediated gene transfer induced leukemia in of 11 children treated for severe combined immunodeficiency raising significant safety issues for gene transfer strategies that cannot be targeted to specific sequences Here, we examine the use of a mobile retroposable genetic element that can be targeted to site specifically introduce therapeutic sequences into mutant genes The data demonstrate that the mobile group II intron from Lactococcus lactis can be targeted to insert into and repair mutant lacZ and ß-globin genes with high efficiency and fidelity in model systems in bacteria These results suggest that these mobile genetic elements represent a novel class of agents for performing targeted genetic repair We are also studying the group II intron’s ability to repair DNA targets in mammalian cells Previous work in our lab has shown that the intron’s functional entity, an RNP, can repair therapeutically relevant targets in 293 and CEM T cells 379 New Zinc Finger Protein Nuclease Architectures for More Efficient Gene Modification Therapies Jeffrey C Miller,1 Lei Zhang,1 Fyodor D Urnov,1 Sarah Patterson,1 Danny Xia,1 Jeremy Rock,1 Sheldon Augustus,1 Carl O Pabo,1 Philip D Gregory,1 Edward J Rebar,1 Michael C Holmes.1 Internal Research, Sangamo BioSciences Inc., Richmond, CA Recent studies have shown that zinc finger protein nucleases (ZFNs) may be used to introduce precise, therapeutically beneficial changes into the genome This approach offers considerable promise for the correction of monogenic diseases, and should also allow the ablation of genes whose expression is harmful in particular disease settings Realizing the full potential of ZFN-mediated gene modification therapy, however, will require the development of methods for routinely engineering ZFNs that cleave with high efficiency at virtually any DNA sequence, since a separation distance of as few as forty bp between the sites of nuclease cleavage and desired sequence change can lead to a significant reduction in gene modification efficiencies In order to address these issues we have engineered and characterized several improvements to the architecture of our designed ZFNs In initial studies, we systematically varied the length and composition of the linker connecting the zinc finger and nuclease domains, and determined the impact of these changes on the cleavage efficiency and optimal target arrangement of the resultant ZFN (which functions as a dimer) It was observed both in vitro and in vivo that ZFNs could be efficiently designed to cleave targets separated by 48 bps, with maximal activity obtained when monomer sites were separated by 4, or bp Next, structure-based design was used to engineer asymmetric contacts into the dimer interface in order to create ZFNs that could function only as heterodimers This was of interest from the standpoint of enhancing both the specificity and efficiency of ZFN performance, since prohibiting formation of the therapeutically irrelevant homodimer would eliminate the possibility of off-target effects Biochemical and cellular studies revealed that the resultant ZFNs functioned as desired Finally, we examined whether re-ordering of the zinc finger and nuclease domains within the ZFN polypeptide would enable functional assembly of novel dimer arrangements on the target DNA (“head to head’ and “head to tail”, in addition to the natural “tail to tail” arrangement) This was of interest because the availability of an increased number of productive arrangements would directly enhance our capacity to design ZFNs to cleave at any chosen base We have demonstrated that the “re-ordered” ZFNs may be combined with each other and with the parental architecture to yield dimers that cut preferentially in all three configurations (“head to head”, “tail to tail” and “head to tail”) Together, these approaches have significantly improved the cleavage efficiency and targeting specificity of the ZFN platform, and have enhanced our capacity to design ZFNs for use in therapeutic gene modification 380 Studies on the Mechanism of Gene Repair Using Saccharomyces cerevisae as a Model System Katie Maguire,1 Hetal Parekh-Olmedo,1 Eric B Kmiec.1 Biological Sciences, University of Delaware, Newark, DE Oligonucleotides are being used to direct base changes within genes and can be used as a gene therapy for human disease While the entire mechanism of the targeted nucleotide exchange (TNE) reaction has yet to be elucidated, there is evidence that it may be separated into at least two phases: the pairing phase, which consists of the MSSO pairing to the target site within the gene, and the repair phase, in which the MSSO directs the repair of the Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy S147 ... there is evidence that it may be separated into at least two phases: the pairing phase, which consists of the MSSO pairing to the target site within the gene, and the repair phase, in which the. .. tail” and “head to tail”) Together, these approaches have significantly improved the cleavage efficiency and targeting specificity of the ZFN platform, and have enhanced our capacity to design ZFNs... procedures is that they themselves trigger accumulation and proliferation of vascular smooth muscle (VSM) cells from the tunica media to tunica intima of the vascular wall leading to restenosis or