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99 Zinc Finger Nuclease Mediated Genomic Editing of CCR5 in Human Hematopoietic Stem Cells for Clinical Application in HIV Gene Therapy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright ©[.]
STEM CELL THERAPIES I 97 LYL1, a Basic Helix-Loop-Helix Transcription Factor, Promotes Hematopoietic Differentiation of Common Marmoset Embryonic Stem Cells Hirotaka Kawano,1 Tomotoshi Marumoto,1,2 Michiyo Okada,1 Tomoko Inoue,1 Takenobu Nii,1 Jiyuan Liao,1 Saori Yamaguchi,1 Yoko Nagai,1 Hiroyuki Inoue,1,2 Atsushi Takahashi,1,2 Erika Sasaki,3 Yoshie Miura,1 Kenzaburo Tani.1,2 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Department of Advanced Molecular and Cell Therapy, Kyushu University Hospital, Fukuoka, Japan; 3Central Institute for Experimental Animals, KEIO University, Kawasaki, Japan Since the successful establishment of human embryonic stem cell (ESC) lines by James Thomson in 1998, transplantation of differentiated ES cells to the specific impaired organ has been expected to cure its defective function For the establishment of the regenerative medicine, the preclinical studies using animal model systems including non-human primates are essential We have recently demonstrated that non-human primate of common marmoset (CM) is suitable for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93: 2839-48, 2009] Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by the ectopic expression of TAL1/SCL However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1/SCL gene is not enough to induce functional HSCs which have self-renewal capability and multipotency Thus we tried to find other hematopoietic genes being able to promote hematopoietic differentiation more efficiently than Tal1/SCL when they are expressed in CM ESCs We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in embryoid bodies(EBs) derived from Cj11, one of CM ESC lines, dramatically increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors We also found that LYL1overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages by CFU (colony-forming unit) assay These results indicate that the ectopic expression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro Furthermore we found that the combined activation of TAL1/SCL and LYL1 can enhance the emergence of CD34+ cells from CM ESCs than the activation of TAL1/SCL or LYL1 alone This technology to induce hematopoietic differentiation of ESCs by the transduction of specific transcription factors are novel, and might be applicable for human regenerative medicine to cure human blood diseases 98 Genome-Wide Definition of Transcriptionally Active Regulatory Elements in Human Stem Cells by Retroviral Scanning Valentina Poletti,1 Alessia Cavazza,2 Annarita Miccio,2 Danilo Pellin,3 Ermanno Rizzi,4 Giorgio Corti,4 Clelia Di Serio,3 Gianluca De Bellis,4 Fulvio Mavilio.1,2 Gene Expression Unit, H.San Raffaele Scientific Institute, Milano, Italy; 2Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy; 3CUSSB, Vita-Salute San Raffaele University, Milano, Italy; 4Institute of Biomedical Technologies, CNR, Milano, Italy The rapidly expanding information on the structural and functional characteristics of the human genome allows the development of genome-wide approaches to the understanding of the molecular Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy circuitry wiring the genetic and epigenetic programs of somatic stem cells High-throughput approaches are essential to study transcriptomes, chromatin dynamics and the use of regulatory elements in the genome Very few studies exist for somatic stem cells, due to the absence of unambiguous markers for their prospective isolation We have recently developed a tool to map functional regions of chromatin in stem and progenitor cells, based on the integration properties of the Moloney murine leukemia virus (MLV) By using a deep sequencing technology, we mapped >32,000 MLV integration sites in the genome of human CD34+ hematopoietic stem/progenitor cells (HPCs), and designed a detailed integration map throughout the genome MLV integrations cluster in regions that co-map with transcriptionally active, cell-specific promoters and regulatory regions bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z) Single locus analysis showed association of MLV clusters with known enhancers and promoters, and allow the identifications of new regions with putative regulatory function (Cattoglio et al., Blood 2010) Functional genome mapping through retroviral “scanning” may be used as a tool to identify regulatory regions involved in the execution of stem cell genetic programs, providing a substantial contribution to a comprehensive description of the molecular circuitry associated with stem cell functions We are exploiting this technique to identify regulatory elements associated with stemness, commitment and differentiation of prospectively isolated or retrospectively defined human stem cells and their progeny, such as HPCs and erythroid and myeloid committed precursors, embryonic stem (ES) cells and ES-derived neural precursors, epidermal stem/progenitor cells (EpSCs) cells and EpSC-derived keratinocytes “Integromes” of at least 30,000 independent integrations are correlated to gene expression profiles and epigenetic genome annotations, and compared by rigorous statistical analysis to discover shared or lineage-specific regulatory regions 99 Zinc Finger Nuclease Mediated Genomic Editing of CCR5 in Human Hematopoietic Stem Cells for Clinical Application in HIV Gene Therapy Lijing Li,1 Anitha Rao,1 Lan-Feng Cao,1 Kenneth Kim,3 Ursula Hofer,2 Paula Cannon,2 Philip D Gregory,3 Michael C Holmes,3 John Zaia,1 Qing Liu,1 John Rossi,1 David DiGiusto.1 Virology, City of Hope National Medical Center, Duarte, CA; University of Southern California, Los Angeles, CA; 3Sangamo BioScience, Richmond, CA The HIV co-receptor CCR5 is a validated target for HIV/AIDS therapy We are currently developing methodologies for ex vivo treatment of hematopoietic stem cells (HSC) with Zinc Finger Nucleases (ZFN) designed to disrupt CCR5 expression with the intent of using these cells for autologous transplantation into patients with AIDS Adenoviral vectors have been developed to deliver and express Green Fluorescence Protein (Ad5/F35-GFP) or a CCR5-specific ZFN (Ad5/F35-SB728) for these studies We compared several compounds (interferon α, pleiotrophin and phorbol 12-myristate 13-acetate PMA) known to have an effect on HSC activation, proliferation and differentiation for their ability to enhance adenoviral transduction, expression and (ultimately) ZFN activity at the CCR5 locus in HSCs CD34+ HSC were isolated from G-CSF mobilized peripheral blood and prestimulated with SCF, Flt-3L and Tpo and then transduced with various amounts of adenovirus or prestimulated (as above) plus either IFNα, PTN, or PMA prior to viral transduction Cells were assayed for viability, GFP expression, ZFN activity and in vitro hematopoietic potential over a week period Our results demonstrate that while as much as 50% of the cells are transduced and express GFP (from Ad5/F35-GFP) when exposed to vector alone, only PMA treatment resulted in both increased frequency of transduction (>98%), protein expression and ZFN activity (using Ad5/F35 vector expressing the S39 CARDIOVASCULAR GENE & CELL THERAPY CCR5 ZFNs) with up to 45% of the genomic CCR5 target sites modified We have established doses of PMA and virus that support high levels of ZFN activity with minimal impact on hematopoietic potential as measured in in vitro assays We also tested endosomal disruption agents (chloroquine and oakadeic acid) for their ability to enhance ZFN expression in this system Both compounds enhanced ZFN activity alone and Chloraquine also enhanced PMA-mediated ZFN activity These results demonstrate that ZFN activity is enhanced in CD34+ cells following PMA treatment at least in part due to induction of very high levels of protein expression and that endosomal disruption further enhances ZFN activity in this system We are currently comparing these conditions in vivo in an immunodeficient mouse model (NSG) to determine the effect of low dose PMA on engraftment and lymphoid lineage potential 100 Human Neural Stem Cells of Parthenogenetic Origin Ruslan Semechkin,1 Dmitry Isaev,1 Tatiana Abramihina,1 Nikolay Turovets,1 Richard A West,2 Tatjana Zogovic-Kapsalis,1 Andrey Semechkin.1 International Stem Cell Corporation, Oceanside, CA; 2Western Michigan University, Kalamazoo, MI The use of multipotent neural stem cells (NSC) in regenerative medicine may provide a potential treatment for Alzheimer’s and Parkinson’s diseases, amyotrophic lateral sclerosis and other neurodegenerative diseases, with human pluripotent stem cells providing a potentially unlimited source of NSC In this study, we demonstrated for the first time that human multipotent NSC stable to long-term passaging can be derived from pluripotent human parthenogenetic stem cells, followed by differentiation into neurons and glial cells Human parthenogenetic stem cells (hpSC) are derived from the inner cell mass of blastocysts obtained from unfertilized oocytes that have been activated using parthenogenesis These cells demonstrate characteristics typical for human embryonic stem cells (hESC), including extensive self-renewal and in vivo as well as in vitro differentiation into cells of all three germ layers Differentiated derivatives of HLA homozygous hpSC with common haplotypes may reduce the risk of immune rejection after transplantation; thus offering significant advantages for application in cell-based therapies due to their enhanced ability to be immune-matched over hESC lines that carry HLA genes in heterozygous form Moreover, derivation of hpSC overcomes ethical hurdles associated with the derivation of hESC from fertilized human embryos For NSC derivation we use an approach based on the adherent model with modifications Rosettes of neuroepithelial cells were formed in the colonies of hpSC grown in the absence of feeder cells The generated rosettes expressed specific neuroepithelial markers such as PAX6, SOX1, NES (Nestin), MSI1 (Musashi-1), and did not express pluripotency marker OCT4 The rosettes were isolated, disaggregated into single cell suspension and then propagated as an adhesion culture The human parthenogenetic NSC (hpNSC) were passaged every 4-5 days enzymatically with a 1:2 split rate for more than 15 passages Reverse transcriptase realtime quantitative PCR (qRT-PCR) revealed the expression of specific neural markers NES (Nestin), SOX2 and MSI1 (Musashi-1) in the hpNSC which was also confirmed by immunocytochemical staining of corresponding proteins At the same time, the expression of OCT4 was not detected at RNA and protein levels The cells keep uniform morphology up to at least 15 passages and not show constantly increasing levels of ectomesenchymal markers SNAI1 and FOXD3 as detected by qRT-PCR To demonstrate the ability to produce neurons and glia, we allowed spontaneous differentiation for three weeks After this time most of the differentiated cells have acquired specific neuron morphology and were positive for anti-Tuj1 (Tubulin beta III) labeled antibodies The expression of neuronal markers TUBB3 (Tubulin beta III) and MAP2 as well as glial markers FOXO4 and S40 GFAP was detected in these differentiated cells by qRT-PCR The ethical and immunomatching advantages make hpSC a very promising source of multipotent NSC for cell-based transplantation therapy On this basis, we derived for a first time hpNSC that can be maintained in culture and expanded for clinical usage Cardiovascular Gene & Cell Therapy 101 Initial Results from a First-in-Man Study Delivering Non-Viral Gene Therapy JVS-100 To Treat Ischemic Heart Failure Marc S Penn,1 Joseph Pastore,2 Rahul Aras,2 Didier Rouy,3 Gary Schaer,4 MaryJane Farr,5 Warren Sherman,5 Farrell Mendelsohn,6 Douglas Losordo.7 Cleveland Clinic Foundation, Cleveland, OH; 2Juventas Therapeutics, Cleveland, OH; 3BioCardia, San Carlos, CA; 4Rush University Medical Center, Chicago, IL; 5Columbia University, New York, NY; 6Cardiology PC, Birmingham, AL; 7Northwestern University, Chicago, IL Stromal cell-derived factor-1 (SDF-1) has been shown to have significant effects in wound healing in multiple organ systems in a variety of animal models, including heart failure (HF) SDF-1 has been shown to recruit bone marrow derived stem cells, cardiac stem cells and to reduce cardiac myocyte cell death JVS-100 is a non-viral plasmid encoding human SDF-1 under control of the CMV promoter We previously demonstrated that JVS-100 was safe at doses up to 100 mg and improved cardiac function and angiogenesis at doses up to 30 mg in a GLP study delivering JVS-100 via the BioCardia Helical Infusion Catheter (HIC) to pigs with ischemic HF We propose to present month results for all cohorts of a Phase open-label study to test safety and bioactivity of JVS-100 in patients (pts) with class III HF due to prior myocardial infarction (MI) Methods and Results: 17 NYHA Class III pts with prior MI (mean time from MI > years) and ejection fraction ≤ 40% were enrolled doses of JVS-100: mg (n=4 pts), 15 mg (n=6 pts), and 30 mg (n=7 pts), were delivered to the peri-MI region via 15 endomyocardial injections with the BioCardia HIC Pts are being followed at 1, 4, and 12 months post-injection Safety is being evaluated by tracking adverse events with the primary safety endpoint being the number of major adverse cardiac events at 30 days Bioactivity is assessed by measuring changes from baseline in: echocardiographic parameters including left ventricular volume and ejection fraction, cardiac perfusion via SPECT imaging at baseline and months after treatment and clinical parameters including NYHA class, minute walk distance and quality of life All pts received 15 injections of JVS-100 To date, pts have been followed for a total of 64 months Follow-up at and months in the mg cohort and month in the 15 mg cohort has been completed There have been no adverse events likely related to the drug at month in the 15 pts who have reached their month follow-up visit At months postinjection, all pts in the mg group improved resting perfusion compared to baseline, demonstrating bioactivity of JVS-100 Using a composite endpoint of clinically significant changes in combining echocardiographic and clinical endpoints, of pts improved in the mg cohort at months and of pts improved at month in the 15 mg cohort month safety and efficacy data will be available for all pts in April 2011 Conclusions: The initial first-in-man data suggests delivery of a non-viral SDF-1 gene therapy, JVS-100, via endomyocardial injection to post-MI heart failure patients is safe JVS-100 demonstrates bioactivity at the mg dose level by improving resting perfusion Follow-up at all dose levels is ongoing and will provide further insight into JVS-100 safety and bioactivity at higher dose levels Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... parthenogenesis These cells demonstrate characteristics typical for human embryonic stem cells (hESC), including extensive self-renewal and in vivo as well as in vitro differentiation into cells of. .. postinjection, all pts in the mg group improved resting perfusion compared to baseline, demonstrating bioactivity of JVS-100 Using a composite endpoint of clinically significant changes in combining echocardiographic... very promising source of multipotent NSC for cell-based transplantation therapy On this basis, we derived for a first time hpNSC that can be maintained in culture and expanded for clinical usage