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496. Optimised Factor VIII Vectors for Gene Therapy of Haemophilia A
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 1 Research and Development, Celsense, Inc., Pittsburgh, PA; 2 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 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 1 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; 3 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
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 1 Research and Development, Celsense, Inc., Pittsburgh, PA; 2 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 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 1 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; 3 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