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Nonviral Vector Development
NONVIRAL VECTOR DEVELOPMENT need to understand the forces responsible for damage during lyophilization in order to develop stable, dehydrated formulations. Our earlier studies have emphasized the role of maintaining particle size in preserving transfection rates during the lyophilization process. In this study, we demonstrate that aggregation typically occurs during the freezing step of the lyophilization process due to the concentration of vectors in the unfrozen fraction. As freezing progresses, vectors become more concentrated and aggregation is promoted. Furthermore, vectors remain in this concentrated solution for 10-24 hours during primary drying. We suggest that excipients can prevent aggregation by isolating individual particles within a sugar matrix that inhibits interactions between particles leading to aggregation. Surprisingly, the formulation of vectors in high concentrations of hydroxyethylstarch is not sufficient to preserve particle size and transfection rates, despite the formation of a glassy matrix. Conversely, glucose preserves particle size at temperatures that do not allow vitrification to occur. Taken together, these results indicate that factors other than the formation of a glassy excipient matrix are responsible for the preservation of nonviral vectors during lyophilization. We will present our current work on the preservation of different nonviral vector types (e.g., lipid-based, polymerbased), and the ability of various excipients to preserve particle size and in vitro transfection rates during lyophilization.
NONVIRAL VECTOR DEVELOPMENT 968. Single Chain Fv: A Ligand in Receptor-mediated Gene Delivery Sanhita Gupta, Jean Eastman, Pamela Davis
We have used an anti-human polyimmunoglobulin receptor (pIgR) single chain Fv (scFv) to deliver reporter genes to epithelial cells in vitro. We have previously demonstrated successful transfection of pIgR-bearing cells in rats with Fab portion of anti-rat pIgR as ligand, which proved immunogenic on repeated administration (Ferkol et al 1995). The scFv ligand was constructed from monoclonal antibodies directed against human pIgR to reduce the species-specific sequences and therefore the immunogenicity of the ligand, and a cys residue was added at the carboxyl end to facilitate its conjugation to polylysine (polyK) via the heterobifunctional cross linker SPDP. ScFv-cys was expressed in Drosophila S2 cells and purified using conventional column chromatography. ScFv-polyK, and polyK as control, were condensed with a DNA expression plasmid containing the luciferase reporter gene under CMV promoter into unimolecular (with respect to DNA) complexes under high salt conditions. Target cells were MDCK cells transfected with human pIgR and repeatedly sorted for high level receptor expression, with untransfected MDCK cells as control. Receptor bearing MDCK cells were readily transfected by scFv containing, pIgR- directed complexes, and expression could be blocked by addition of excess human secretory component, the extracellular portion of pIgR. In contrast, MDCK cells that did not express pIgR were not transfected. Non targeted complexes were not effective in transfecting MDCK cells with or without pIgR. These data indicate that an scFv directed against human pIgR can direct foreign genes specifically into receptor-bearing cells in vitro. We speculate that such a ligand, like the Fab tested previously, will deliver foreign genes into pIgR bearing cells in vivo, but with less immunogenicity. This study was supported by NIH grants DK27651, DK49138, The Cystic Fibrosis Foundation grant HL07415 and Copernicus Therapeutics, Inc.
969. The Nuclear Uptake of Lactosylated Polylysine/DNA Complex in CF Cells
Daniel Klink, Mary Catherine Glick, Thomas F. Scanlin The Cystic Fibrosis Center and Department of Pediatrics, University of Pennsylvania School of Medicine, The Childrens Hospital of Philadelphia, Philadelphia, PA, 19104 Lactosylated polylysine was used to transfer the CFTR gene into CF airway epithelial cells and correct the chloride channel dysMOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
function (1). Polylysine, with 40% of the amino-groups substituted with lactose, is also an effective vector to transfer reporter genes into CF and non-CF airway epithelial cells in vitro with a 90-100 % efficiency (2), comparable to that of viral vectors. However, little is known about the intracellular route taken by the complex. FITC-labeled lactosylated polylysine was complexed to rhodamine-labeled pCMVLacZ in a 4:1 (w:w) ratio. CF airway epithelial cells were grown overnight on cover slips and subsequently transfected at 37°C. Fixation took place at various times after administration of the transfection medium. A Leica TCS4D confocal microscope was used for the optical sectioning of the fixed cells. Nuclei were visualized with Hoechst stain. Final images were composed by digitally overlaying the images of the different fluorophores. Internalization took place rapidly and ten minutes after administration the lactosylated polylysine/plasmid complex was detected around the nucleus. Distinct nuclear localization of the complex was present one hour after administration. At the same time some dissociation of the plasmid and the vector was observed. After six hours, the complex accumulated mainly in the perinuclear region as well as in the nucleus. Therefore the transport from the cytoplasm into the nucleus may be the rate limiting step in this process. The rapid internalization is indicative of uptake by receptor mediated endocytosis. The colocalization of the plasmid and the lactosylated polylysine in the perinuclear region allows us to hypothesize a chaperone function for the vector and that a nuclear targeting signal may be involved. These results show that the complex is taken up and transported rapidly through the cytoplasm to the nucleus in toto. The presence of the lactosylated polylysine/plasmid complex was clearly observed in the nucleus, however further characterization is needed to elucidate the specific intracellular path taken by the lactosylated polylysine/plasmid complex to the nucleus. Supported by NIH R21DK55610-01 1. Kollen WJW, Mulberg AE, Wei X, Sugita M, Raghuram V, Wang J. Foskett JK, Glick MC and Scanlin TF, 1999. Hum. Gene Therapy. 10, 615-622. 2. Kollen WJW, Schembri FM, Gerwig GJ, Vliegenthart JFG, Glick MC and Scanlin TF, 1999. Am J. Respir. Cell. Mol. Biol. 20, 1081-1086.
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970. Retroviral Transduction of CD341 Cells on Fibronectin Fragment CH-296 Is Inhibited by High Concentrations of Virus Particles Present in the Medium— Implications for Design of Gene Transfer Protocols for Hematopoietic Cells Thomas Relander, Stefan Karlsson, Johan Richter Molecular Medicine and Gene Therapy, University of Lund, Sweden
Objective: To develop a simple but efficient protocol for gene transfer to human CD341 and CD341/CD38- using oncoretroviral vectors and to compare transduction efficiency of three differently pseudotyped vectors. Methods: An MSCV based vector with the gene for enhanced green fluorescent protein (MGIN) was packaged into three different packaging cell lines: GP1envAM12 (amphotropic envelope), PG13 (GALV envelope) and 293GPG (VSV-G envelope). Viral titer was assessed on HT1080 cells for each batch of harvested virus and found to be . 2x106 infectious particles/ml. Cord blood CD341 cells were pre-stimulated in serum free medium in the presence of TPO, FL and SCF (100ng/ml) for 48 h and then transduced once on Retronectin. Results: The transduction efficiency obtained with pre-load (PL) of virus onto Retronectin alone was compared to pre-load plus addition of virus containing medium (VCM):
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NONVIRAL VECTOR DEVELOPMENT 968. Single Chain Fv: A Ligand in Receptor-mediated Gene Delivery Sanhita Gupta, Jean Eastman, Pamela Davis
We have used an anti-human polyimmunoglobulin receptor (pIgR) single chain Fv (scFv) to deliver reporter genes to epithelial cells in vitro. We have previously demonstrated successful transfection of pIgR-bearing cells in rats with Fab portion of anti-rat pIgR as ligand, which proved immunogenic on repeated administration (Ferkol et al 1995). The scFv ligand was constructed from monoclonal antibodies directed against human pIgR to reduce the species-specific sequences and therefore the immunogenicity of the ligand, and a cys residue was added at the carboxyl end to facilitate its conjugation to polylysine (polyK) via the heterobifunctional cross linker SPDP. ScFv-cys was expressed in Drosophila S2 cells and purified using conventional column chromatography. ScFv-polyK, and polyK as control, were condensed with a DNA expression plasmid containing the luciferase reporter gene under CMV promoter into unimolecular (with respect to DNA) complexes under high salt conditions. Target cells were MDCK cells transfected with human pIgR and repeatedly sorted for high level receptor expression, with untransfected MDCK cells as control. Receptor bearing MDCK cells were readily transfected by scFv containing, pIgR- directed complexes, and expression could be blocked by addition of excess human secretory component, the extracellular portion of pIgR. In contrast, MDCK cells that did not express pIgR were not transfected. Non targeted complexes were not effective in transfecting MDCK cells with or without pIgR. These data indicate that an scFv directed against human pIgR can direct foreign genes specifically into receptor-bearing cells in vitro. We speculate that such a ligand, like the Fab tested previously, will deliver foreign genes into pIgR bearing cells in vivo, but with less immunogenicity. This study was supported by NIH grants DK27651, DK49138, The Cystic Fibrosis Foundation grant HL07415 and Copernicus Therapeutics, Inc.
969. The Nuclear Uptake of Lactosylated Polylysine/DNA Complex in CF Cells
Daniel Klink, Mary Catherine Glick, Thomas F. Scanlin The Cystic Fibrosis Center and Department of Pediatrics, University of Pennsylvania School of Medicine, The Childrens Hospital of Philadelphia, Philadelphia, PA, 19104 Lactosylated polylysine was used to transfer the CFTR gene into CF airway epithelial cells and correct the chloride channel dysMOLECULAR THERAPY Vol. 1, No. 5, May 2000, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
function (1). Polylysine, with 40% of the amino-groups substituted with lactose, is also an effective vector to transfer reporter genes into CF and non-CF airway epithelial cells in vitro with a 90-100 % efficiency (2), comparable to that of viral vectors. However, little is known about the intracellular route taken by the complex. FITC-labeled lactosylated polylysine was complexed to rhodamine-labeled pCMVLacZ in a 4:1 (w:w) ratio. CF airway epithelial cells were grown overnight on cover slips and subsequently transfected at 37°C. Fixation took place at various times after administration of the transfection medium. A Leica TCS4D confocal microscope was used for the optical sectioning of the fixed cells. Nuclei were visualized with Hoechst stain. Final images were composed by digitally overlaying the images of the different fluorophores. Internalization took place rapidly and ten minutes after administration the lactosylated polylysine/plasmid complex was detected around the nucleus. Distinct nuclear localization of the complex was present one hour after administration. At the same time some dissociation of the plasmid and the vector was observed. After six hours, the complex accumulated mainly in the perinuclear region as well as in the nucleus. Therefore the transport from the cytoplasm into the nucleus may be the rate limiting step in this process. The rapid internalization is indicative of uptake by receptor mediated endocytosis. The colocalization of the plasmid and the lactosylated polylysine in the perinuclear region allows us to hypothesize a chaperone function for the vector and that a nuclear targeting signal may be involved. These results show that the complex is taken up and transported rapidly through the cytoplasm to the nucleus in toto. The presence of the lactosylated polylysine/plasmid complex was clearly observed in the nucleus, however further characterization is needed to elucidate the specific intracellular path taken by the lactosylated polylysine/plasmid complex to the nucleus. Supported by NIH R21DK55610-01 1. Kollen WJW, Mulberg AE, Wei X, Sugita M, Raghuram V, Wang J. Foskett JK, Glick MC and Scanlin TF, 1999. Hum. Gene Therapy. 10, 615-622. 2. Kollen WJW, Schembri FM, Gerwig GJ, Vliegenthart JFG, Glick MC and Scanlin TF, 1999. Am J. Respir. Cell. Mol. Biol. 20, 1081-1086.
ADDITION
TO
STEM CELL
AND
BLOOD POSTER SESSION
970. Retroviral Transduction of CD341 Cells on Fibronectin Fragment CH-296 Is Inhibited by High Concentrations of Virus Particles Present in the Medium— Implications for Design of Gene Transfer Protocols for Hematopoietic Cells Thomas Relander, Stefan Karlsson, Johan Richter Molecular Medicine and Gene Therapy, University of Lund, Sweden
Objective: To develop a simple but efficient protocol for gene transfer to human CD341 and CD341/CD38- using oncoretroviral vectors and to compare transduction efficiency of three differently pseudotyped vectors. Methods: An MSCV based vector with the gene for enhanced green fluorescent protein (MGIN) was packaged into three different packaging cell lines: GP1envAM12 (amphotropic envelope), PG13 (GALV envelope) and 293GPG (VSV-G envelope). Viral titer was assessed on HT1080 cells for each batch of harvested virus and found to be . 2x106 infectious particles/ml. Cord blood CD341 cells were pre-stimulated in serum free medium in the presence of TPO, FL and SCF (100ng/ml) for 48 h and then transduced once on Retronectin. Results: The transduction efficiency obtained with pre-load (PL) of virus onto Retronectin alone was compared to pre-load plus addition of virus containing medium (VCM):
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