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836. Stable Correction of a Genetic Defect by In Vivo Targeting of a Stem Cell Population with a Lentiviral Vector
GENE THERAPY OF MUSCLE DISEASES high-level expression of mini-agrin protein in injected muscle. More importantly, H&E staining showed that mini-agrin positive area has a significant muscle morphology improvement compare with agrin negative area. Collagen III staining also demonstrated less fibrosis occurred in agrin positive area. To achieve better therapeutic effect, systemic delivery of mini-agrin protein to the whole body of lama2dy mouse by AAV vector is underway. This study will provide a potential treatment for CMD.
836. Stable Correction of a Genetic Defect by In Vivo Targeting of a Stem Cell Population with a Lentiviral Vector Gary P. Kobinger,1,2 Jean-Pierre Louboutin,1,2 Elizabeth R. Barton,1 H. Lee Sweeney,1 James M. Wilson.1,2 1 University of Pennsylvania School of Medicine, Philadelphia, PA; 2 The Wistar Institute, Philadelphia, PA. Successful gene replacement therapy will require stability of the genetic graft or therapeutic gene in the corrected tissues. Stability of the genetic graft can be assured with integrating or self replicating viruses by targeting post-mitotic cells that have a very long life-time or stem cells that can replenish defective tissue with corrected cells. Most differentiated cells that are targets of gene transfer turnover with frequencies that could require repeated delivery of vector or vector modified cells at intervals ranging from weeks to months. An alternative strategy is to genetically modify stem cells capable of stably repopulating diseased tissues. This has been achieved in several models in which stem cells are genetically modified ex vivo prior to transplantation. In this study, we explore the possibility of targeting a stem cell population in situ through in vivo administration of vector. For developing this concept, we have selected the mouse model of muscular dystrophy (mdx mice) that undergo rapid turnover of their muscle fibers. Results reveal that in vivo targeting of muscle stem cells, notably satellite cells, with a pseudotyped lentiviral vector encoding for the minidystrophin restores dystrophin expression and provides functional correction in skeletal muscle of mdx mice. Targeting of satellite cells in mdx mice with Ebola or MuLV pseudotyped HIV vector expressing dystrophin results in the regeneration of functionally and phenotypically corrected mature skeletal muscles. This study shows that progenitor cells can be genetically engineered in vivo and subsequently proliferate into terminally differentiated tissue carrying the genetic graft in a way that stably corrects function. James M. Wilson holds equity in Targeted Genetics, Corp.
MOLECULAR CONJUGATES 837. Tumor Specific Gene Delivery Mediated by a Novel Peptide/Polyethylenimine/DNA Polyplex Targeting Aminopeptidase N (CD13) Stanley Saamoah-Moffatt,1 Sandra Wiehle,1 Richard J. Cristiano.1 1 Department of Genitourinary Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, United States. The ability to obtain specific and efficient gene delivery remains as one of the greatest limitations of cancer gene therapy. This is especially critical in the development of delivery vectors that are designed to target tumors following systemic administration. In contrast to many current non-viral vectors that result in low delivery to tumors, we have developed a novel polyethylenimine(PEI)/DNA vector formulation that is capable of efficient delivery following an intravenous administration in that there is greater delivery to tumor than lungs. We demonstrate here, that the specificity of delivery mediated by this novel vector can be increased even further by S322
attaching a peptide specific for aminopeptidase N (CD13) to the vector. The CD13/Aminopeptidase N-binding NGR peptide (CNGRC) (isolated by phage-display technology) was utilized based on its ability to target tumor and tumor endothelial cells. This peptide was coupled to our novel vector by attaching phenyl(di)boronic acid (PDBA) to the peptide via a polyethylene glycol (PEG) linker and salicylhydroxamic acid (SHA) to PEI, allowing for a selfassembling linkage between PDBA and SHA. FACS analysis identified the highest expression of CD13 on primary human umbilical vein endothelial cells (HUVEC) and the human tumor cell lines UC3 (bladder), HT-1080 (fibrosarcoma), H1299 (lung), and PC3-MM2 (Prostate) cells, while little or no expression was apparent on C4-2 (Prostate), MCF-7 (Breast), and LnCapD (Prostate) cells. In vitro assessment of targeting by the CNGRC/PEG/PEI/DNA vector carrying a βgal expressing plasmid on CD13 positive H1299, HT1080, UC-3 and HUVEC cells showed as much as a 5-fold increase in transduction over the untargeted PEG/PEI/DNA vector. To confirm specificity, competition with a 100-fold molar excess of free peptide resulted in up to a 90% reduction in delivery while little or no increase in transduction was identified on the CD13 negative LnCapD cell line transduced with the targeted vector, indicating that gene delivery was specific for CD13 positive cells. Intravenous administration of the CNGRC/PEG/PEI/DNA vector to nude mice bearing H1299 subcutaneous tumors resulted in as much as a 12fold increase in βgal expression in tumors as compared to βgal expression in either lungs or tumors from control treated animals. In vivo transduction analysis using the CNGRC/PEG/PEI/DNA vector to target the intravenous delivery of a yellow fluorescence protein (YFP) expressing plasmid to subcutaneous H1299 tumors confirmed delivery to both tumor cells and tumor vasculature. The use of this peptide to increase the tumor specific delivery mediated by our novel PEI/DNA vector now provides a basis for developing tumor targeted gene therapies for use in the clinical treatment of cancer.
838. Liver-Targeted Gene Transfer Mediated by Chitosan-DNA Nanoparticles through Bile Duct, Portal Vein and Tail Vein Injections H. Dai,1,3 P. C. Zhang,1 W. S. Lim,1 Y. Chen,3 K. W. Leong,1,2 H. Q. Mao.1,2 1 Tissue and Therapeutics Engineering Lab, Johns Hopkins Singapore, Singapore; 2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, United States; 3 Department of Hepatobiliary Surgery, Xijing Hospital, the Fourth Military Medical University, Xian, China. Non-viral gene therapy has significant clinical potential, but its therapeutic utility is still hindered by a low transfection efficiency due to a combination of extracellular and intracellular barriers. The gene transfer efficiency can be improved by optimization of gene carriers and administration routes. For instance, liver-targeted gene transfer, in principle, can be improved by localized administration of the DNA nanoparticles. We have previously reported that chitosan-DNA nanoparticles can effect successful gene transfer following oral administration. In this study, we examine the feasibility of achieving liver-targeted gene delivery by chitosan-DNA nanoparticles through bile duct and portal vein infusions. The duration of transgene expression in rat liver following administration of chitosan-DNA nanoparticles was compared with that of PEIDNA nanoparticles or naked DNA. Chitosan-DNA nanoparticles (N/P=3), PEI-DNA nanoparticles (N/P=10) or naked DNA containing 200 mg of plasmid encoding firefly luciferase (Vical VR1255) in 4 mL of medium were infused into the common bile duct of Wistar rats (0.2 mL/min) or portal vein (2 mL/min) using a syringe pump. Tail vein injection was performed using a 33 gauge needle over 2 min, as a control. At days 3, 7 and 14, five rats from each group were sacrificed and the luciferase activity in tissue extracts Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
836. Stable Correction of a Genetic Defect by In Vivo Targeting of a Stem Cell Population with a Lentiviral Vector Gary P. Kobinger,1,2 Jean-Pierre Louboutin,1,2 Elizabeth R. Barton,1 H. Lee Sweeney,1 James M. Wilson.1,2 1 University of Pennsylvania School of Medicine, Philadelphia, PA; 2 The Wistar Institute, Philadelphia, PA. Successful gene replacement therapy will require stability of the genetic graft or therapeutic gene in the corrected tissues. Stability of the genetic graft can be assured with integrating or self replicating viruses by targeting post-mitotic cells that have a very long life-time or stem cells that can replenish defective tissue with corrected cells. Most differentiated cells that are targets of gene transfer turnover with frequencies that could require repeated delivery of vector or vector modified cells at intervals ranging from weeks to months. An alternative strategy is to genetically modify stem cells capable of stably repopulating diseased tissues. This has been achieved in several models in which stem cells are genetically modified ex vivo prior to transplantation. In this study, we explore the possibility of targeting a stem cell population in situ through in vivo administration of vector. For developing this concept, we have selected the mouse model of muscular dystrophy (mdx mice) that undergo rapid turnover of their muscle fibers. Results reveal that in vivo targeting of muscle stem cells, notably satellite cells, with a pseudotyped lentiviral vector encoding for the minidystrophin restores dystrophin expression and provides functional correction in skeletal muscle of mdx mice. Targeting of satellite cells in mdx mice with Ebola or MuLV pseudotyped HIV vector expressing dystrophin results in the regeneration of functionally and phenotypically corrected mature skeletal muscles. This study shows that progenitor cells can be genetically engineered in vivo and subsequently proliferate into terminally differentiated tissue carrying the genetic graft in a way that stably corrects function. James M. Wilson holds equity in Targeted Genetics, Corp.
MOLECULAR CONJUGATES 837. Tumor Specific Gene Delivery Mediated by a Novel Peptide/Polyethylenimine/DNA Polyplex Targeting Aminopeptidase N (CD13) Stanley Saamoah-Moffatt,1 Sandra Wiehle,1 Richard J. Cristiano.1 1 Department of Genitourinary Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, United States. The ability to obtain specific and efficient gene delivery remains as one of the greatest limitations of cancer gene therapy. This is especially critical in the development of delivery vectors that are designed to target tumors following systemic administration. In contrast to many current non-viral vectors that result in low delivery to tumors, we have developed a novel polyethylenimine(PEI)/DNA vector formulation that is capable of efficient delivery following an intravenous administration in that there is greater delivery to tumor than lungs. We demonstrate here, that the specificity of delivery mediated by this novel vector can be increased even further by S322
attaching a peptide specific for aminopeptidase N (CD13) to the vector. The CD13/Aminopeptidase N-binding NGR peptide (CNGRC) (isolated by phage-display technology) was utilized based on its ability to target tumor and tumor endothelial cells. This peptide was coupled to our novel vector by attaching phenyl(di)boronic acid (PDBA) to the peptide via a polyethylene glycol (PEG) linker and salicylhydroxamic acid (SHA) to PEI, allowing for a selfassembling linkage between PDBA and SHA. FACS analysis identified the highest expression of CD13 on primary human umbilical vein endothelial cells (HUVEC) and the human tumor cell lines UC3 (bladder), HT-1080 (fibrosarcoma), H1299 (lung), and PC3-MM2 (Prostate) cells, while little or no expression was apparent on C4-2 (Prostate), MCF-7 (Breast), and LnCapD (Prostate) cells. In vitro assessment of targeting by the CNGRC/PEG/PEI/DNA vector carrying a βgal expressing plasmid on CD13 positive H1299, HT1080, UC-3 and HUVEC cells showed as much as a 5-fold increase in transduction over the untargeted PEG/PEI/DNA vector. To confirm specificity, competition with a 100-fold molar excess of free peptide resulted in up to a 90% reduction in delivery while little or no increase in transduction was identified on the CD13 negative LnCapD cell line transduced with the targeted vector, indicating that gene delivery was specific for CD13 positive cells. Intravenous administration of the CNGRC/PEG/PEI/DNA vector to nude mice bearing H1299 subcutaneous tumors resulted in as much as a 12fold increase in βgal expression in tumors as compared to βgal expression in either lungs or tumors from control treated animals. In vivo transduction analysis using the CNGRC/PEG/PEI/DNA vector to target the intravenous delivery of a yellow fluorescence protein (YFP) expressing plasmid to subcutaneous H1299 tumors confirmed delivery to both tumor cells and tumor vasculature. The use of this peptide to increase the tumor specific delivery mediated by our novel PEI/DNA vector now provides a basis for developing tumor targeted gene therapies for use in the clinical treatment of cancer.
838. Liver-Targeted Gene Transfer Mediated by Chitosan-DNA Nanoparticles through Bile Duct, Portal Vein and Tail Vein Injections H. Dai,1,3 P. C. Zhang,1 W. S. Lim,1 Y. Chen,3 K. W. Leong,1,2 H. Q. Mao.1,2 1 Tissue and Therapeutics Engineering Lab, Johns Hopkins Singapore, Singapore; 2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, United States; 3 Department of Hepatobiliary Surgery, Xijing Hospital, the Fourth Military Medical University, Xian, China. Non-viral gene therapy has significant clinical potential, but its therapeutic utility is still hindered by a low transfection efficiency due to a combination of extracellular and intracellular barriers. The gene transfer efficiency can be improved by optimization of gene carriers and administration routes. For instance, liver-targeted gene transfer, in principle, can be improved by localized administration of the DNA nanoparticles. We have previously reported that chitosan-DNA nanoparticles can effect successful gene transfer following oral administration. In this study, we examine the feasibility of achieving liver-targeted gene delivery by chitosan-DNA nanoparticles through bile duct and portal vein infusions. The duration of transgene expression in rat liver following administration of chitosan-DNA nanoparticles was compared with that of PEIDNA nanoparticles or naked DNA. Chitosan-DNA nanoparticles (N/P=3), PEI-DNA nanoparticles (N/P=10) or naked DNA containing 200 mg of plasmid encoding firefly luciferase (Vical VR1255) in 4 mL of medium were infused into the common bile duct of Wistar rats (0.2 mL/min) or portal vein (2 mL/min) using a syringe pump. Tail vein injection was performed using a 33 gauge needle over 2 min, as a control. At days 3, 7 and 14, five rats from each group were sacrificed and the luciferase activity in tissue extracts Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy