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650. Gold Nanoparticle Mediated Gene Delivery Via Surface Charge Reversal with Pyridinium Amphiphiles
GENE & CELL THERAPY FOR HEMOPHILIA AND NOVEL TECHNICAL APPROACHES lipids. Moreover, we have found that positively charged pyridinium polar head can confer a good DNA binding/release profile due to its soft charge, delocalized on the six-member ring system [1]. In the present study a series of pyridinium gemini surfactants bearing hydrophilic linkers was synthesized from a set of reactive lipophilic pyrylium salts and 1,5-diamino-3-oxapentane, following our original procedure previously reported [1]. Hydrophobic chains of the new surfactants were elongated from 10 to 17 carbon atoms for testing the influence of this structural element on the amphiphile self-assembling properties. We also undertook a counterion study, analyzing the impact of counterion properties (hydration, lipophilicity) on the self-assembling process. The self-assembling and physicochemical properties were studied in bulk via DSC and in solution, through nanoDSC and aggregation experiments. We have found that the self-assembling of the novel pyridinium amphiphiles is significantly affected by both the chain length and counterion (as expected) and that these physicochemical properties can be directly correlated with the transfection efficiency of the vectors, assessed on NCI-H23 lung cancer cell line. The transfection efficiency can be further enhanced by using zwitterionic lipids such as DOPE, while formulations containing cholesterol were less efficient. The complete structureactivity relationship will be presented, with pertinent physicochemical and self-assembling data. Optimized formulations based on the new pyridinium amphiphiles were able to transfect the same cancer cell line in the presence of elevated levels of serum, mimicking the in vivo transfection conditions and showing great promise for in vivo use. [1] Ilies, M.A., et al., Lipophilic pyrylium salts in the synthesis of efficient pyridinium-based cationic lipids, gemini surfactants, and lipophilic oligomers for gene delivery. J. Med. Chem., 2006. 49(13), 3872-3887.
650. Gold Nanoparticle Mediated Gene Delivery Via Surface Charge Reversal with Pyridinium Amphiphiles
Adrian Kizewski,1 Marc A. Ilies.1 1 Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA. Efficient delivery of nucleic acids for therapeutic uses requires multifunctional delivery systems able to compact the genetic material, provide targeting, while being able to be tracked efficiently in vivo. Among various platforms available, gold nanoparticles were recently gaining momentum due to their availability, biocompatibility, density, and special physicochemical, optical and surface properties. The study presents our recent efforts to generate an efficient gene delivery system based on gold nanoparticles decorated with pyridinium positivelycharged thiols and (partially) stabilized with thiolated ethyleneglycol oligomers. The fine tuning of relevant physicochemical properties of the nanosystems (size, charge, nature and density of ligands) will be shown, emphasizing their impact towards the stability, transfection efficiency and cytotoxicity of the novel nanodelivery platform.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
Gene & Cell Therapy for Hemophilia and Novel Technical Approaches 651. A Novel iPSC-Based Strategy To Correct the Bleeding Phenotype in Hemophilia A
Gabriella Ranaldo,1 Yvonne Richaud-Patin,2 Angelo Lombardo,3 Chantal Grosso,1 Maria Talmon,1 Angel Raya,2 Luigi Naldini,3 Piercarla Schinco,4 Antonia Follenzi.1 1 University of Piemonte Orientale, Novara, Italy; 2Inst. for Bioengineering of Catalonia, Barcelona, Spain; 3HSR_TIGET, Milano, Italy; 4Az. Osp/Univ S.Giovanni Battista-Molinette, Torino, Italy. Hemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. Currently, there is no definitive cure. Therefore, cell and gene therapy may represent powerful solutions for the permanent treatment of HA. Reprogramming of genetically corrected somatic cells can be used to generate high amount of autologous, disease-free induced Pluripotent Stem Cells (iPSC), which can be then differentiated into progenitor cells relevant for gene and cell therapy applications. Towards this goal, we first generated iPSC from human fibroblasts derived from healthy donors by retroviral transduction with four factors (OCT4, KLF4, SOX2 and c-MYC). These cells were phenotypically similar to human embryonic stem cells (hESC): colonies were compact, uniform and with defined borders when grown on feeder cells and expressed specific stem cell markers such as alkaline phosphatase, Nanog, Oct3/4, Sox2, TRA-1-60, TRA-1-81 and SSEA 3/4. iPSC were competent for differentiation into cell types of the three germ layers. Importantly, iPSC differentiated into endothelial cells (EC), a cell type that, when transplanted in HA mice, allows correcting the hemorrhagic phenotype of this model. iPSC can differentiate into EC acquiring a typical endothelial-like morphology with increased expression of CD31, KDR and FVIII. Moreover, after differentiation these cells were amenable to gene transfer by LV expressing the clotting human B-domain-deleted FVIII (hBDD-FVIII) under control of an endothelial-specific VE-cadherin promoter. Using this method we obtained iPSC, but they cannot be used for future therapeutic approach for the risk of reactivation of the reprogramming genes in the iPSC progeny. Thus, we recently reprogrammed human and mouse fibroblasts with a Cre-excisable LV expressing OCT4, KLF4 and SOX2. One month after transduction, colonies displayed a hESC-like morphology and stained positive for embryonic stem cell markers. RT-PCR and WB analyses showed activation of the endogenous reprogramming factors in iPSC. Given these results, we reprogrammed HA mouse fibroblasts into iPSC both before and after correction with a LV expressing hBDD-FVIII under control of the PGK promoter. Importantly, corrected iPSC expressed detectable BDD-FVIII in reprogrammed cells that were differentiated in EC. However, in hemophilic patients, to harvest fibroblasts from skin biopsies is risky; for this reason, we utilized peripheral blood cells as an easy-to-access source of cells and reprogrammed mononuclear cells from donors and hemophilic patients with HA. After genetic correction with LV and Cre-mediated excision of the reprogramming vector, the iPSC will be differentiated into EC and transplanted into NOD-SCID HA mice. Overall, these data will be instrumental to assess the engraftment, the proliferation and the levels of FVIII expression from differentiated, gene corrected and reprogramming factor free iPSC to confirm the suitability of this approach for hemophilia gene-cell-therapy.
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650. Gold Nanoparticle Mediated Gene Delivery Via Surface Charge Reversal with Pyridinium Amphiphiles
Adrian Kizewski,1 Marc A. Ilies.1 1 Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA. Efficient delivery of nucleic acids for therapeutic uses requires multifunctional delivery systems able to compact the genetic material, provide targeting, while being able to be tracked efficiently in vivo. Among various platforms available, gold nanoparticles were recently gaining momentum due to their availability, biocompatibility, density, and special physicochemical, optical and surface properties. The study presents our recent efforts to generate an efficient gene delivery system based on gold nanoparticles decorated with pyridinium positivelycharged thiols and (partially) stabilized with thiolated ethyleneglycol oligomers. The fine tuning of relevant physicochemical properties of the nanosystems (size, charge, nature and density of ligands) will be shown, emphasizing their impact towards the stability, transfection efficiency and cytotoxicity of the novel nanodelivery platform.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
Gene & Cell Therapy for Hemophilia and Novel Technical Approaches 651. A Novel iPSC-Based Strategy To Correct the Bleeding Phenotype in Hemophilia A
Gabriella Ranaldo,1 Yvonne Richaud-Patin,2 Angelo Lombardo,3 Chantal Grosso,1 Maria Talmon,1 Angel Raya,2 Luigi Naldini,3 Piercarla Schinco,4 Antonia Follenzi.1 1 University of Piemonte Orientale, Novara, Italy; 2Inst. for Bioengineering of Catalonia, Barcelona, Spain; 3HSR_TIGET, Milano, Italy; 4Az. Osp/Univ S.Giovanni Battista-Molinette, Torino, Italy. Hemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. Currently, there is no definitive cure. Therefore, cell and gene therapy may represent powerful solutions for the permanent treatment of HA. Reprogramming of genetically corrected somatic cells can be used to generate high amount of autologous, disease-free induced Pluripotent Stem Cells (iPSC), which can be then differentiated into progenitor cells relevant for gene and cell therapy applications. Towards this goal, we first generated iPSC from human fibroblasts derived from healthy donors by retroviral transduction with four factors (OCT4, KLF4, SOX2 and c-MYC). These cells were phenotypically similar to human embryonic stem cells (hESC): colonies were compact, uniform and with defined borders when grown on feeder cells and expressed specific stem cell markers such as alkaline phosphatase, Nanog, Oct3/4, Sox2, TRA-1-60, TRA-1-81 and SSEA 3/4. iPSC were competent for differentiation into cell types of the three germ layers. Importantly, iPSC differentiated into endothelial cells (EC), a cell type that, when transplanted in HA mice, allows correcting the hemorrhagic phenotype of this model. iPSC can differentiate into EC acquiring a typical endothelial-like morphology with increased expression of CD31, KDR and FVIII. Moreover, after differentiation these cells were amenable to gene transfer by LV expressing the clotting human B-domain-deleted FVIII (hBDD-FVIII) under control of an endothelial-specific VE-cadherin promoter. Using this method we obtained iPSC, but they cannot be used for future therapeutic approach for the risk of reactivation of the reprogramming genes in the iPSC progeny. Thus, we recently reprogrammed human and mouse fibroblasts with a Cre-excisable LV expressing OCT4, KLF4 and SOX2. One month after transduction, colonies displayed a hESC-like morphology and stained positive for embryonic stem cell markers. RT-PCR and WB analyses showed activation of the endogenous reprogramming factors in iPSC. Given these results, we reprogrammed HA mouse fibroblasts into iPSC both before and after correction with a LV expressing hBDD-FVIII under control of the PGK promoter. Importantly, corrected iPSC expressed detectable BDD-FVIII in reprogrammed cells that were differentiated in EC. However, in hemophilic patients, to harvest fibroblasts from skin biopsies is risky; for this reason, we utilized peripheral blood cells as an easy-to-access source of cells and reprogrammed mononuclear cells from donors and hemophilic patients with HA. After genetic correction with LV and Cre-mediated excision of the reprogramming vector, the iPSC will be differentiated into EC and transplanted into NOD-SCID HA mice. Overall, these data will be instrumental to assess the engraftment, the proliferation and the levels of FVIII expression from differentiated, gene corrected and reprogramming factor free iPSC to confirm the suitability of this approach for hemophilia gene-cell-therapy.
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