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1103. Site-Specific Chromosomal Integration in Human Mesenchymal Stem Cells Mediated by PhiC31 Integrase
MOLECULAR CONJUGATES: VECTOR ENGINEERING electroporation using the Amaxa nucleoporator. One plasmid carrying an enhanced Green Fluorescent Protein (eGFP) reporter cassette (to facilitate FACS analysis of resultant cells) flanked by the SB IR sequences for permanent integration into the target cell chromosomes was co-nucleoporated with a second plasmid carrying the SB transposase expression cassette. We first used the K562 human erythro-leukemia cell line to optimize several parameters of gene delivery, achieving high levels (50-80%) of stable gene integration with optimal amounts and ratios of the two plasmids. Southern analysis of K562 clonal populations showed that 1 to 6 copies of the transposon can be detected. We then nucleoporated the SBbased system into human CD34+ progenitor cells isolated from umbilical cord blood and achieved initial gene transfer to these primary cells at moderate levels (10-30%) that led to stable expression of the reporter gene (1-4%). Results from these studies demonstrate the potential for using the SB transposon system for gene therapy using HSC.
1103. Site-Specific Chromosomal Integration in Human Mesenchymal Stem Cells Mediated by PhiC31 Integrase Annahita Keravala,1 Juergen Hoelters,2 Sohail Jarrahian,1 Thomas W. Chalberg,1 Marisa Ciccarella,2 Peter Neth,2 Michele P. Calos.1 1 Genetics, Stanford University School of Medicine, Stanford, CA; 2 Division of Clinical Chemistry and Clinical Biochemistry at the Department of Surgery, City Center, Ludwig-MaximiliansUniversity, Munich, Germany. Human mesenchymal stem cells (hMSCs) have emerged as a promising new tool in gene therapy/ cell therapy strategies due to their potential use in a wide variety of applications, including both short- and long-term regeneration. With viral vectors being frequently criticized for their immunogenicity, random integration and difficult production, alternatives are desirable. A safe and effective gene delivery method resulting in targeted chromosomal integration with prolonged therapeutic levels of gene expression would be valuable. PhiC31 integrase, a site-specific recombinase from phage phiC31, pairs an attB recognition site on an incoming plasmid with pseudo attP sites in mammalian genomes. This integrase has been used to achieve genomic integration in human keratinocytes, mouse liver, rat retina and rabbit joints. Here we describe non-viral approaches for gene delivery using phiC31 integrase, transfected either as mRNA or as a DNA expression vector, in conjugation with an attB-containing donor plasmid. We evaluated the efficacy of mRNA encoding phiC31 integrase to mediate unidirectional integration into pseudo attP sites when co-transfected with an attB donor plasmid containing a transgene of interest. We also demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the luciferase marker gene and an attB site resulted in 39-fold higher expression of luciferase as assessed by live imaging, compared to control transfections without integrase. Expansion of the integrants and their differentiation into the osteogenic phenotype revealed sustained gene expression, indicating that the integration sites used in undifferentiated hMSCs continued to provide gene expression after differentiation of the cells. Finally, plasmid rescue and DNA sequence analysis of the integration sites showed that the integration events occurred at several pseudo attP sites in the human genome. Summarizing our results, phiC31 integrase, provided either as mRNA or DNA, mediates site-specific integration of transgenes and can provide high levels of gene expression in hMSCs and differentiated osteogenic cells. These findings suggest that phiC31 integrase represents an efficient and practical non-viral approach for genetic modification of hMSCs that could have potential for clinical applications.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
MOLECULAR CONJUGATES: VECTOR ENGINEERING 1104. Imaging of sec-R Directed and PEGStabilized Gene Transfer Nanoparticles in CF Mice Maxwell Kotlarchyk,1 Zhenghong Lee,1 Mark Cooper,2 Pamela B. Davis,1 Assem G. Ziady.1 1 Pediatrics, Case Western Reserve University, Cleveland, OH; 2 Copernicus Therapeutics, Inc., Cleveland, OH. Compacted DNA targeted to the serpin enzyme complex receptor (sec-R) can deliver amounts of human CFTR DNA sufficient for partial correction of the chloride transport defect and NOS-2 downregulation in the noses of CF knock out mice. To examine the distribution of these compacted DNA particles in vivo, we used radioscintigraphy imaging. We modified our DNA compacting agent (a polymer of poly-L-lysine, chemically conjugated to the C105Y sec-R targeting ligand) with radiolabeled I-125. Prior to dosing, 12 S489X/FABP-hCFTR were imaged by X-ray for proper anatomical alignment with radiograph images. Following complex formulation, 9 mice were dosed intranasally (IN) with 25 µg of sec-R targeted radiolabeled hCFTR DNA (specific activity 50 µCi each), while 3 control mice received administration of free I-125 (specific activity 50 µCi each). We used a small animal gamma scanner to analyze radio images at 2, 4, 24, and 48 hrs following nasal administration of the dose. At each time 3 animals were sacrificed, frozen and stored for subsequent sectioning. For each mouse, I-125 scintigraphic images, x-ray images, and autoradiographs were aligned and analyzed. Targeted delivery resulted in higher total retained activity than the carrier control. Both test and control groups begin to exhibit thyroid uptake by 2 hrs, while significantly higher activities were observed in the nasal and lower airways for animals that received the targeted complexes vs. controls. The highest observed activity was in thyroid, liver and bladder. Autoradiographic images correlated well with their corresponding in vivo scintigraphic images. For mice that received targeted complexes, strong signals were measured in both abdominal (stomach and liver) and bladder regions indicating possible ingestion during or aspiration following administration, as well as secretion into the bladder. This rapid clearance by 2 hrs was observed for all mice. Regional analysis showed significantly (p<0.05) higher activities for the targeted delivery in nose, lung and liver vs. controls, 2 and 4 hrs post administration. By 24 hrs, activities in the upper airways and lungs were back to the background level, while activity in the thyroid remained significant at 24 and 48 hrs. The data indicate a rapid clearance of sec-R targeted compacted DNA (by 2 hrs), followed by the appearance of at least the labeled component of the complex (the ligand) in the thyroid and bladder by 4 hrs. We also examined expression conferred by second generation PEG-stabilized compacted nanoparticles containing the Firefly luciferase gene using bioluminescent imaging. Expression was detected in lung 2 and 4 days following intra-tracheal (IT) but not IN administration. Lung homogenates from both methods of administration exhibited luciferase activity although levels in the IT group were significantly higher. These data suggest that a threshold level of expression, achieved only with IT administration, is necessary for successful bioluminescent detection . Development of these real-time imaging techniques has allowed us to better assess the localization and site of activity of our gene transfer complex formulations. Drs. Ziady and Davis hold equity and consult for Copernicus Therapeutics, Inc., a biotech company that has licensed technologies used in this work from Case Western Reserve University.
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1103. Site-Specific Chromosomal Integration in Human Mesenchymal Stem Cells Mediated by PhiC31 Integrase Annahita Keravala,1 Juergen Hoelters,2 Sohail Jarrahian,1 Thomas W. Chalberg,1 Marisa Ciccarella,2 Peter Neth,2 Michele P. Calos.1 1 Genetics, Stanford University School of Medicine, Stanford, CA; 2 Division of Clinical Chemistry and Clinical Biochemistry at the Department of Surgery, City Center, Ludwig-MaximiliansUniversity, Munich, Germany. Human mesenchymal stem cells (hMSCs) have emerged as a promising new tool in gene therapy/ cell therapy strategies due to their potential use in a wide variety of applications, including both short- and long-term regeneration. With viral vectors being frequently criticized for their immunogenicity, random integration and difficult production, alternatives are desirable. A safe and effective gene delivery method resulting in targeted chromosomal integration with prolonged therapeutic levels of gene expression would be valuable. PhiC31 integrase, a site-specific recombinase from phage phiC31, pairs an attB recognition site on an incoming plasmid with pseudo attP sites in mammalian genomes. This integrase has been used to achieve genomic integration in human keratinocytes, mouse liver, rat retina and rabbit joints. Here we describe non-viral approaches for gene delivery using phiC31 integrase, transfected either as mRNA or as a DNA expression vector, in conjugation with an attB-containing donor plasmid. We evaluated the efficacy of mRNA encoding phiC31 integrase to mediate unidirectional integration into pseudo attP sites when co-transfected with an attB donor plasmid containing a transgene of interest. We also demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the luciferase marker gene and an attB site resulted in 39-fold higher expression of luciferase as assessed by live imaging, compared to control transfections without integrase. Expansion of the integrants and their differentiation into the osteogenic phenotype revealed sustained gene expression, indicating that the integration sites used in undifferentiated hMSCs continued to provide gene expression after differentiation of the cells. Finally, plasmid rescue and DNA sequence analysis of the integration sites showed that the integration events occurred at several pseudo attP sites in the human genome. Summarizing our results, phiC31 integrase, provided either as mRNA or DNA, mediates site-specific integration of transgenes and can provide high levels of gene expression in hMSCs and differentiated osteogenic cells. These findings suggest that phiC31 integrase represents an efficient and practical non-viral approach for genetic modification of hMSCs that could have potential for clinical applications.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
MOLECULAR CONJUGATES: VECTOR ENGINEERING 1104. Imaging of sec-R Directed and PEGStabilized Gene Transfer Nanoparticles in CF Mice Maxwell Kotlarchyk,1 Zhenghong Lee,1 Mark Cooper,2 Pamela B. Davis,1 Assem G. Ziady.1 1 Pediatrics, Case Western Reserve University, Cleveland, OH; 2 Copernicus Therapeutics, Inc., Cleveland, OH. Compacted DNA targeted to the serpin enzyme complex receptor (sec-R) can deliver amounts of human CFTR DNA sufficient for partial correction of the chloride transport defect and NOS-2 downregulation in the noses of CF knock out mice. To examine the distribution of these compacted DNA particles in vivo, we used radioscintigraphy imaging. We modified our DNA compacting agent (a polymer of poly-L-lysine, chemically conjugated to the C105Y sec-R targeting ligand) with radiolabeled I-125. Prior to dosing, 12 S489X/FABP-hCFTR were imaged by X-ray for proper anatomical alignment with radiograph images. Following complex formulation, 9 mice were dosed intranasally (IN) with 25 µg of sec-R targeted radiolabeled hCFTR DNA (specific activity 50 µCi each), while 3 control mice received administration of free I-125 (specific activity 50 µCi each). We used a small animal gamma scanner to analyze radio images at 2, 4, 24, and 48 hrs following nasal administration of the dose. At each time 3 animals were sacrificed, frozen and stored for subsequent sectioning. For each mouse, I-125 scintigraphic images, x-ray images, and autoradiographs were aligned and analyzed. Targeted delivery resulted in higher total retained activity than the carrier control. Both test and control groups begin to exhibit thyroid uptake by 2 hrs, while significantly higher activities were observed in the nasal and lower airways for animals that received the targeted complexes vs. controls. The highest observed activity was in thyroid, liver and bladder. Autoradiographic images correlated well with their corresponding in vivo scintigraphic images. For mice that received targeted complexes, strong signals were measured in both abdominal (stomach and liver) and bladder regions indicating possible ingestion during or aspiration following administration, as well as secretion into the bladder. This rapid clearance by 2 hrs was observed for all mice. Regional analysis showed significantly (p<0.05) higher activities for the targeted delivery in nose, lung and liver vs. controls, 2 and 4 hrs post administration. By 24 hrs, activities in the upper airways and lungs were back to the background level, while activity in the thyroid remained significant at 24 and 48 hrs. The data indicate a rapid clearance of sec-R targeted compacted DNA (by 2 hrs), followed by the appearance of at least the labeled component of the complex (the ligand) in the thyroid and bladder by 4 hrs. We also examined expression conferred by second generation PEG-stabilized compacted nanoparticles containing the Firefly luciferase gene using bioluminescent imaging. Expression was detected in lung 2 and 4 days following intra-tracheal (IT) but not IN administration. Lung homogenates from both methods of administration exhibited luciferase activity although levels in the IT group were significantly higher. These data suggest that a threshold level of expression, achieved only with IT administration, is necessary for successful bioluminescent detection . Development of these real-time imaging techniques has allowed us to better assess the localization and site of activity of our gene transfer complex formulations. Drs. Ziady and Davis hold equity and consult for Copernicus Therapeutics, Inc., a biotech company that has licensed technologies used in this work from Case Western Reserve University.
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