- Email: [email protected]
464. Hematopoietic Differentiation of Airway Epithelial Cell-Derived Cystic Fibrosis (CF) Induced Pluripotent Stem (iPS) Cells
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II isolated and serially transplanted to Fah mutant mice in order to prove the stable integration of the siRNA constructs. We identified two TAT and one HPD siRNA constructs that could partially rescue Fah mutant mice. The mice injected with siRNAs showed improved liver functions and reduced body weight loss during NTBC withdrawal. Mice serially transplanted with hepatocytes from these mice also showed more tolerance to NTBC withdrawal and improved liver function. Conclusion: shRNA knockdown of TAT and HPD can render hepatocytes resistant to Fah deficiency and can serve in vivo selection genes.
464. Hematopoietic Differentiation of Airway Epithelial Cell-Derived Cystic Fibrosis (CF) Induced Pluripotent Stem (iPS) Cells
R. Geoffrey Sargent,1,5 Yanu Yang,5 Albert Lee,1,5 Marcus O. Muench,6 Soya Kim,5 Arash Bedayat,5 Layli Jamali,5 Lin Ye,3 Judy Chang,3 Dieter C. Gruenert.1,2,4,5,7 1 Otolaryngology-Head and Neck Surgery, University of California, San Francisco, CA; 2Laboratory Medicine, University of California, San Francisco, CA; 3Medicine, University of California, San Francisco, CA; 4Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Helen Diller Family Comprehensive Cancer Center, Institute for Human Genetics, University of California, San Francisco, CA; 5Cell Biology, California Pacific Medical Center Research Institute, San Francisco, CA; 6Blood Systems Research Institute, Department of Laboratory Medicine and Liver Center, University of California, San Francisco, CA; 7Medicine, University of Vermont, Burlington, VT. CF is the most common inherited disease in the Caucasian population and has significant multiorgan damage that appears to compromise both epithelial and inflammatory cell function. The development of induced pluripotent stem (iPS) cells is a significant advance in the development of cell-based therapy that could benefit inherited diseases such as CF. The iPS cell system has the potential for generating patient-specific pluripotent stem (PS) cells that can ultimately be differentiated into tissue-specific precursor cells for repair of tissues and organs affected by the disease pathologies. CF-iPS cells were derived from airway epithelial cells of a patient with a ∆F508/∆F508 genotype. These cells have been characterized for their pluripotent features including the expression of antigens associated with pluripotence (Nanog, SSEA3, SSEA4, Tra-1-60, Tra1-81), as well as the ability to recapitulate the 3 primary germ layers (mesoderm, ectoderm, and endoderm) in vitro in embryoid bodies and in vivo as teratomas. Because CF pathology appears to manifest in inflammatory cells as well as epithelial cells, studies were undertaken to determine whether the CF epithelial cells could be differentiated into hematopoietic/inflammatory cell precursors. CF-iPS cells were grown under defined conditions and analyzed by FACs. The FACS analysis indicated the presence and co-expression of leukosialin (CD43) and the common leukocyte antigen, CD45 (CD43+CD45+) in a distinct subpopulation derived from the differentiation of the CF-iPS cells. Gene correction studies and kinetic analysis will be used to determine the corrected precursor population that gives rise to this more differentiated inflammatory cells population. This work is supported by funds from Cystic Fibrosis Research, Inc and Pennsylvania Cystic Fibrosis, Inc and NIH grant GM75111.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
465. Quantitation of Transgene Expression with Isotope Dilution Mass Spectrometry
Michael J. Passineau,1 Kalyan Paila,2 Ramaz Geguchadze,1 H. M. Skip Kingston.2 1 Cardiovascular Medicine, Allegheny-Singer Research Institute, Pittsburgh, PA; 2Chemistry, Duquesne University, Pittsburgh, PA.
Gene transfer studies are dosed according to a paradigm that assumes a constant relationship between the number of vector units delivered and the number of protein effector (transgene) molecules produced. Thus, gene therapy studies usually attempt to directly correlate physiological response to vector dose administered. Whereas this methodology has generally proven adequate for preclinical and even clinical gene therapy studies, an improved system would be one wherein vector dose, effector molecules expressed, and physiological endpoints could all be known with precision. Quantitation of transgene effector molecules may be very difficult, since many gene therapies are designed for diseases wherein the genetic defect is not a deletion but rather a mutation, meaning that the mutated endogenous protein may be virtually indistinguishable from the transgene effector molecule with immunological methods. This is of particular importance for our interest in gene therapy for Fabry disease, where required levels of the α-Galactosidase A (GLA) therapeutic are low, and difficult to distinguish from mutated endogenous GLA. We have designed a strategy, based upon Stable Isotope Dilution Mass Spectrometry (SIDMS) for protein quantitation that involves modifying the C-terminus of the GLA transgene with a strep-tag, allowing us to spike the sample with a known quantity of a strep-tagged isotopically enriched synthetic peptide mix that mimic the transgene as an internal standard and to co-purify the transgene; and the internal standard from a lysate of mouse liver following hydrodynamic gene delivery. This method is useful as a proof-of-principle due to the availability of the strep-tag for affinity purification. The concept could be extended to modify the C-terminus and/or N-terminus of a therapeutic transgene by the addition of as few as one amino acid, resulting in a mass shift of the tryptic amino acid sequence of the protein. In our example, tryptic digestion of GLA-strep-tag yields a C-terminal 24-amino acid peptide of mass 2733.4 Da. The isotopically-enriched internal standard, also a 24-amino acid (AA) C-terminal peptide with a mass upshift of +8 Da and total mass of 2741.4 Da. Both the 2733.4 Dalton peak and the 2741.4 Dalton peak were clearly identified by SIDMS and the 24-AA tryptic sequence tag was validated by Tandem MS or MS/MS spectrum of the mouse liver spiked with the isotopically enriched internal standard, allowing us precise quantitation of our strep-tagged GLA transgene.
465.5 Direct Gene Transfer with Compacted DNA Nanoparticles in Retinal Pigment Epithelial Cells: Expression, Repeat Delivery and Lack of Toxicity
Zongchao Han,1 Adarsha Koirala,1 Rasha Makkia,1 Mark J. Cooper,2 Muna I. Naash.1 1 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Copernicus Therapeutics Inc., Cleveland, OH.
The goal of this study is to evaluate the efficacy and safety profiles of single and multiple subretinal deliveries of compacted DNA nanoparticles (NPs). EGFP expression cassettes transcriptionallycontrolled by the Vitelliform Macular Dystrophy 2 (VMD2) promoter were constructed as linear (L-eGFP) and circular (C-eGFP) DNA and compacted into rod-shaped neutral-charged NPs having a diameter of 8-11 nm in size using polyethylene glycol-substituted lysine 30-mers. The VMD2 promoter was used to specifically target retinal pigment epithelial (RPE) cells. Single or double 1µl subretinal injections of NPs or naked DNA (both at 4.3 µg DNA/ml) were performed at postnatal (P) day 30 BALB/c mice, with the second injection at P60. S179
464. Hematopoietic Differentiation of Airway Epithelial Cell-Derived Cystic Fibrosis (CF) Induced Pluripotent Stem (iPS) Cells
R. Geoffrey Sargent,1,5 Yanu Yang,5 Albert Lee,1,5 Marcus O. Muench,6 Soya Kim,5 Arash Bedayat,5 Layli Jamali,5 Lin Ye,3 Judy Chang,3 Dieter C. Gruenert.1,2,4,5,7 1 Otolaryngology-Head and Neck Surgery, University of California, San Francisco, CA; 2Laboratory Medicine, University of California, San Francisco, CA; 3Medicine, University of California, San Francisco, CA; 4Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Helen Diller Family Comprehensive Cancer Center, Institute for Human Genetics, University of California, San Francisco, CA; 5Cell Biology, California Pacific Medical Center Research Institute, San Francisco, CA; 6Blood Systems Research Institute, Department of Laboratory Medicine and Liver Center, University of California, San Francisco, CA; 7Medicine, University of Vermont, Burlington, VT. CF is the most common inherited disease in the Caucasian population and has significant multiorgan damage that appears to compromise both epithelial and inflammatory cell function. The development of induced pluripotent stem (iPS) cells is a significant advance in the development of cell-based therapy that could benefit inherited diseases such as CF. The iPS cell system has the potential for generating patient-specific pluripotent stem (PS) cells that can ultimately be differentiated into tissue-specific precursor cells for repair of tissues and organs affected by the disease pathologies. CF-iPS cells were derived from airway epithelial cells of a patient with a ∆F508/∆F508 genotype. These cells have been characterized for their pluripotent features including the expression of antigens associated with pluripotence (Nanog, SSEA3, SSEA4, Tra-1-60, Tra1-81), as well as the ability to recapitulate the 3 primary germ layers (mesoderm, ectoderm, and endoderm) in vitro in embryoid bodies and in vivo as teratomas. Because CF pathology appears to manifest in inflammatory cells as well as epithelial cells, studies were undertaken to determine whether the CF epithelial cells could be differentiated into hematopoietic/inflammatory cell precursors. CF-iPS cells were grown under defined conditions and analyzed by FACs. The FACS analysis indicated the presence and co-expression of leukosialin (CD43) and the common leukocyte antigen, CD45 (CD43+CD45+) in a distinct subpopulation derived from the differentiation of the CF-iPS cells. Gene correction studies and kinetic analysis will be used to determine the corrected precursor population that gives rise to this more differentiated inflammatory cells population. This work is supported by funds from Cystic Fibrosis Research, Inc and Pennsylvania Cystic Fibrosis, Inc and NIH grant GM75111.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
465. Quantitation of Transgene Expression with Isotope Dilution Mass Spectrometry
Michael J. Passineau,1 Kalyan Paila,2 Ramaz Geguchadze,1 H. M. Skip Kingston.2 1 Cardiovascular Medicine, Allegheny-Singer Research Institute, Pittsburgh, PA; 2Chemistry, Duquesne University, Pittsburgh, PA.
Gene transfer studies are dosed according to a paradigm that assumes a constant relationship between the number of vector units delivered and the number of protein effector (transgene) molecules produced. Thus, gene therapy studies usually attempt to directly correlate physiological response to vector dose administered. Whereas this methodology has generally proven adequate for preclinical and even clinical gene therapy studies, an improved system would be one wherein vector dose, effector molecules expressed, and physiological endpoints could all be known with precision. Quantitation of transgene effector molecules may be very difficult, since many gene therapies are designed for diseases wherein the genetic defect is not a deletion but rather a mutation, meaning that the mutated endogenous protein may be virtually indistinguishable from the transgene effector molecule with immunological methods. This is of particular importance for our interest in gene therapy for Fabry disease, where required levels of the α-Galactosidase A (GLA) therapeutic are low, and difficult to distinguish from mutated endogenous GLA. We have designed a strategy, based upon Stable Isotope Dilution Mass Spectrometry (SIDMS) for protein quantitation that involves modifying the C-terminus of the GLA transgene with a strep-tag, allowing us to spike the sample with a known quantity of a strep-tagged isotopically enriched synthetic peptide mix that mimic the transgene as an internal standard and to co-purify the transgene; and the internal standard from a lysate of mouse liver following hydrodynamic gene delivery. This method is useful as a proof-of-principle due to the availability of the strep-tag for affinity purification. The concept could be extended to modify the C-terminus and/or N-terminus of a therapeutic transgene by the addition of as few as one amino acid, resulting in a mass shift of the tryptic amino acid sequence of the protein. In our example, tryptic digestion of GLA-strep-tag yields a C-terminal 24-amino acid peptide of mass 2733.4 Da. The isotopically-enriched internal standard, also a 24-amino acid (AA) C-terminal peptide with a mass upshift of +8 Da and total mass of 2741.4 Da. Both the 2733.4 Dalton peak and the 2741.4 Dalton peak were clearly identified by SIDMS and the 24-AA tryptic sequence tag was validated by Tandem MS or MS/MS spectrum of the mouse liver spiked with the isotopically enriched internal standard, allowing us precise quantitation of our strep-tagged GLA transgene.
465.5 Direct Gene Transfer with Compacted DNA Nanoparticles in Retinal Pigment Epithelial Cells: Expression, Repeat Delivery and Lack of Toxicity
Zongchao Han,1 Adarsha Koirala,1 Rasha Makkia,1 Mark J. Cooper,2 Muna I. Naash.1 1 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Copernicus Therapeutics Inc., Cleveland, OH.
The goal of this study is to evaluate the efficacy and safety profiles of single and multiple subretinal deliveries of compacted DNA nanoparticles (NPs). EGFP expression cassettes transcriptionallycontrolled by the Vitelliform Macular Dystrophy 2 (VMD2) promoter were constructed as linear (L-eGFP) and circular (C-eGFP) DNA and compacted into rod-shaped neutral-charged NPs having a diameter of 8-11 nm in size using polyethylene glycol-substituted lysine 30-mers. The VMD2 promoter was used to specifically target retinal pigment epithelial (RPE) cells. Single or double 1µl subretinal injections of NPs or naked DNA (both at 4.3 µg DNA/ml) were performed at postnatal (P) day 30 BALB/c mice, with the second injection at P60. S179