497 Human Erythropoietin Gene Delivery Using an Arginine Grafted Bioreducible Polymer System Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy[.]
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II macrophage, GEMM=granulocyte, erythroid, macrophage, megakaryocyte) after 14 days of culture in differentiation media Figure 2: MRI imaging of cells and persistence after impantation in a rat hindlimb A Proton image of rat hindlimb anatomy B 19F image of implanted labeled cells (acquisition time ∼1hr) C 19F/1H overlay, with 19F rendered in pseudocolor D 19F/1H overlay using a post-sacrifice, 11-hour 19F scan 496 Optimised Factor VIII Vectors for Gene Therapy of Haemophilia A 495 Validation of Cellular Function and Imaging Capability of CD34+ Stem Cells Labeled with a 19F MRI Contrast Agent Brooke Helfer,1 Anthony Balducci,1 Zhina Sadeghi,2 Chris Flask,3 Amy Wesa.1 Research and Development, Celsense, Inc., Pittsburgh, PA; Department of Urology, Case Western Reserve University, Cleveland, OH; 3Depts of Radiology and Biological Engineering, Center for Imaging Research, Case Western Reserve University, Cleveland, OH Non-invasive detection and tracking of stem cell post-delivery lends insightful information on the mechanism of action of an intended therapy and/or possible reasons for therapeutic failure However, rigorous examination of the effects of the contrast agent on the cells in question must be performed to ensure minimal disruption to the therapy In this work, we examine the effects of a novel selfdelivering 19F MRI contrast agent on hematopoietic stem cells (HSC) Comparison of 19F-labeled HSC and unlabeled control cohorts in in vitro colony forming assays resulted in equal numbers of total colony forming units (CFU), as well as individual CFU types, indicating that label presence did not effect multipotency (see Figure 1) In vivo reconstitution studies in mice, using labeled and unlabeled murine bone marrow HSC, resulted in equivalent development of CFU in the spleen, as well as reconstitution of the lymphoid and myeloid compartments The fact that these highly complex processes remain largely unaltered in the presence of contrast agent provides strong evidence of the inertness of the reagent and that the therapeutic potential of the cells is likely maintained Further, proof of principle MR imaging of injected and implanted HSC in a rat thigh was performed, indicating that HSC can be detected with MR methods with the use of this contrast agent The data suggests that imaging data on the location and persistence of delivered CD34+ HSC can be obtained without influencing the function and/or differentiation potential of the therapeutic cells Figure 1: Average of two independent counts of colony formation and types (BFU= Blast forming unit, E=erythroid, GM=granulocyte, S192 Natalie J Ward,1 Suzanne M K Buckley,1 Ahad A Rahim,1 John H McVey,2 Simon N Waddington.1 Gene Transfer Technology Group, Institute for Women’s Health, UCL, London, United Kingdom; 2Thrombosis Research Institute, London, United Kingdom Haemophilia A is a compelling candidate for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagulation factor level, response to treatment can be easily monitored, and FVIII expression can be mediated by many cell types in vivo B-domain deleted (BDD) FVIII protein retains full procoagulant function and is expressed at higher levels than wild type FVIII However a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch (N6) has been previously reported to increase in vitro secretion of FVIII tenfold compared to BDD-FVIII Previously, our group has shown that expression of FVIII protein from a codon optimised cDNA sequence increases expression in comparison to the wild type sequence over 30-fold in vivo yielding over 200% normal human FVIII levels in neonatal mice after injection of a SIN lentiviral vector A longstanding goal for treatment of haemophilia A using gene therapy is to maintain sustained production of FVIII in the absence of an immune response in adult mice In recent studies we have used an optimised vector system including our codon-optimised FVIII N6 cDNA sequence under control of the liver specific promoter LP1 to maximise expression in hepatocytes Further to this, we have also included target sequences for the hematopoietic specific microRNA, miR-142-3p, to eliminate off target expression in antigen presenting cells With this approach we hope to achieve stable long term expression of FVIII in adult FVIII knock-out mice 497 Human Erythropoietin Gene Delivery Using an Arginine-Grafted Bioreducible Polymer System Youngsook Lee,1 Hye Yeong Nam,1 Jaesung Kim,1 Minhyung Lee,1,2 James W Yockman,1 Sug Kyun Shin,3 Sung Wan Kim.1,2 Center for Controlled Chemial Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT; 2Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea; Division of Nephrology, Department of Internal Medicine, NHIC, Ilsan Hospital, Gyeonggi-do, Republic of Korea Anemia is a major complication of chronic kidney disease (CKD) and is primarily due to impaired erythropoietin (EPO) production by the failing kidney, leading to EPO deficiency Erythropoiesis stimulating agents are widely used to treat anemia for CKD and cancer, however, several clinical limitations impede their effectiveness To overcome many of these clinical hurdles, gene therapy providing continuous release has been suggested as an attractive alternative to current intermittently administered erythropoiesis-stimulating agents Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ENGINEERING OF GENE REGULATION Recently, we developed an arginine-grafted bioreducible poly(CBADAH, disulfide amine) [ABP] polymer for non-viral polymer-based gene delivery Here, we extended our previous in vitro studies by evaluating the erythropoietic effect of a single systemic ABP polymerbased human erythropoietin plasmid DNA (phEPO) delivery system on hematocrit level, reticulocyte count, plasma hEPO protein levels, and organ distribution of hEPO mRNA in rats Systemic non-viral gene therapy has been impeded by the low levels of transfection efficiency and lack of sustained gene expression In the present study, we found that a single systemic injection of ABP-complexed phEPO, sustained higher hematocrit levels, increased reticulocytosis, raised expression levels of plasma hEPO In addition, we observed that the distinct temporal and spatial distribution of phEPO/ABP polyplexes contributed to increased erythropoietic effects compared to those of traditional EPO therapies Overall, Our studies demonstrate that the ABP polymer may be used as an advanced carrier for hEPO gene delivery, and provide a potent and attractive clinical approach to enhance erythropoiesis in vivo 498 Development of In Vivo Gene Therapy of Hemophilia A Mice by Intra-Bone Marrow Injection of Lentiviral Vectors Containing a B-Domain Variant of Human Factor VIII Xuefeng Wang,1 Andy Chiang,1 Peiqing Ye,1 Carol H Miao.1,2 Seattle Children’s Hospital Research Institute, Seattle, WA; Department of Pediatrics, University of Washington, Seattle, WA Our previous studies confirmed that hematopoietic stem cells (HSCs) in bone marrow (BM) of hemophilia A (HemA) mice can be efficiently transduced to express the transgene one week after direct intra-bone marrow (iBM) injection of lentiviral vectors (LVs) containing a gene encoding GFP (GFP-LV) or B-domain variant human factor VIII (hFVIII/N6) (E-FVIII-LV) driven by ubiquitous promoters However, the transgene expression levels changed over time GFP+HSC cell numbers in BM reached largest on day after injection of GFP-LV, and then fell quickly afterwards The plasma FVIII levels (0.1-2 U/mL) in E-FVIII-LV-treated mice fluctuated over time and decreased to undetectable one month postinjection; in some mice, anti-FVIII antibodies (Abs) were produced In order to achieve persistent transgene expression, in this study, we combined iBM injection of LVs with immunomodulation Firstly, we investigated if transient depletion of cytotoxic CD8+ T cells by intraperitoneal (I.P.) injection of antiCD8α Ab can prevent the elimination of the LV-transduced cells We injected 0.08 mg antiCD8α Ab per mouse on day -1 and 0.05 mg antiCD8α Ab per mouse on day 4, 11, 16 and 21, and GFP-LV (9.3 × 108 TU/mouse) on day 0, and tracked GFP expression in HSCs in the BM Compared with LV-only treated mice, there were more GFP+HSCs in the BM of LV + antiCD8α Ab treated mice on day (529±65 cells per × 106 BM cells v.s 198±118 cells per × 106 BM cells) Although GFP+HSC number in these two types of mice decreased over time, LV + antiCD8α Ab treated mice still had relatively larger numbers of GFP+HSC on day 63 (38±33 cells per × 106 BM cells v.s 5±4 cells per × 106 BM cells) Flow cytometry data further showed that during antiCD8α Ab treatment, over 80% of CD3+CD8+ cells in BM were depleted on day 3, however, these cells gradually recovered on day and reached to the normal level on day 15 Thus, transient depletion of CD3+CD8+ cells in BM by I.P injection of antiCD8α Ab could partly rescue the transduced BM cells Secondly, we employed 10 HemA/Foxp3-Tg mice in which all T cells over-expressed Foxp3 as an immunosuppressive mouse model and treated them with low (3.5 × 105 TU/mouse) or high (1.7 × 106 TU/ mouse) dosage of E-FVIII-LVs, respectively None of the 10 treated HemA/Foxp3-Tg produced anti-FVIII Abs; however plasma FVIII levels in all 10 mice fell to low levels one month after treatment Thus, increase of CD4+Foxp3+Tregs did not inhibit the elimination of transduced cells by cytotoxic T cells despite that it prevented antiMolecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy FVIII Ab production Based on the above results, in order to achieve persistent transgene expression of hFVIII in the HemA mice treated with iBM injection of E-FVIII-LVs, we will continue to develop more efficient protocols to inhibit cytotoxic-T-cell-mediated elimination of the transduced cells and to expand FVIII-specific Treg cell populations to block anti-FVIII Ab formation simultaneously Engineering of Gene Regulation 499 Upregulation of the PluripotencyAssociated miRNA 302-367 Cluster Using Engineered Transcription Activator-Like Effector (TALE) Activators Morgan L Maeder,1,2 James F Angstman,1 Sam J Linder,1 Deepak Reyon,1 J Keith Joung.1,2 Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA; 2Biological and Biomedical Sciences Program, Harvard University, Boston, MA microRNAs (miRNAs) play critical roles in gene expression, development and pluripotency Certain families of miRNAs are highly expressed in embryonic stem cells (ESCs) and have been shown to play a role in regulating pluripotency For example, recent work has shown that the miRNA 302-367 cluster is a direct target of Oct4, Sox2 and Nanog and plays a critical role in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) Transcription activator-like effectors (TALEs) are DNA-binding proteins from the Xanthomonas genus of bacteria that can be engineered to bind novel DNA sequences Engineered TALEs can be fused to activation domains and act as artificial transcription factors to increase expression of endogenous genes in human cells However, to our knowledge, TALE activators have only been used to stimulate expression of protein coding genes and not of miRNAs Here, we describe the generation of engineered TALE-activators targeted to the miRNA 302-367 promoter region Using single TALE-activators, we observed modest increases in expression of the miRNA 302367 cluster in primary human fibroblasts By expressing multiple TALE activators targeted to neighboring sequences simultaneously, we were able to induce larger increases in miRNA 302-367 cluster expression, presumably due to synergistic transcriptional activation We tested the use of both plasmid DNA and in vitro transcribed mRNA to express TALE-activators in human cells and investigated the duration of elevated miRNA expression over time Our results demonstrate the feasibility of using engineered TALE activators to upregulate expression of an endogenous miRNA cluster The capability to purposefully alter miRNAs levels should provide a useful tool for exploring their critical roles in gene expression, development and pluripotency and for affecting the expression of downstream gene targets Such tools may also enable manipulation of complex phenotypes, such as reprogramming, which require changes in the expression level of hundreds of genes regulated by miRNAs 500 Light-Inducible Spatiotemporal Gene Regulation Using Engineered Transcription Factors Lauren R Polstein,1 Charles A Gersbach.1 Biomedical Engineering, Duke University, Durham, NC Systems that facilitate the precise control of gene expression have numerous applications in the field of gene therapy Current methods control gene expression via constitutive, tissue-specific, or molecularly-induced promoters or by the expression of a genespecific transcriptional activator However, these systems have not been designed to facilitate simultaneous spatial and temporal control of gene expression, which is desirable for many gene therapy and S193 ...ENGINEERING OF GENE REGULATION Recently, we developed an arginine- grafted bioreducible poly(CBADAH, disulfide amine) [ABP] polymer for non-viral polymer- based gene delivery Here, we extended... effect of a single systemic ABP polymerbased human erythropoietin plasmid DNA (phEPO) delivery system on hematocrit level, reticulocyte count, plasma hEPO protein levels, and organ distribution... polymer may be used as an advanced carrier for hEPO gene delivery, and provide a potent and attractive clinical approach to enhance erythropoiesis in vivo 498 Development of In Vivo Gene Therapy of