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636. Development of Non-Viral Gene Delivery Particles Based on the Dynein Light Chain Rp3
CHEMICAL AND MOLECULAR CONJUGATES II 636. Development of Non-Viral Gene Delivery Particles Based on the Dynein Light Chain Rp3
Marianna T. P. Favaro,1 Marcelo A. S. de Toledo,1 Rafael F. Alves,2 Lucimara G. de la Torre,3 Anete P. Souza,1 Adriano R. Azzoni.2 1 Molecular Biology and Genetic Engineering Center, State University of Campinas, Campinas, Brazil; 2Chemical Engineering Department, University of São Paulo, Brazil; 3Faculty of Chemical Engineering, State University of Campinas, Campinas, Brazil. One of the major challenges faced by gene therapy and DNA vaccination is the development of a delivery vector that is both efficient and safe. Although much more effective, viral vectors still raise several concerns about its safety. In this context “artificial viruses” are interesting options, since they are non-viral vectors intended to explore the cell’s architecture in an efficient way, to overcome a series of physical, enzymatic and diffusional barriers, while still preserving the safety of plasmid DNA (pDNA) vectors. The main objective of this work is to exploit molecular motors, like dynein, to transport cargoes from the periphery to the centrosome of mammalian cells via the microtubule network. For that, human dynein light chain Rp3 was fusioned to a N-terminal DNA binding domain and a C-terminal membrane active peptide, TAT. The protein, named TAT-RD4, has additionally a HisTag. The shuttle protein built contains therefore different domains to promote pDNA condensation (DNA binding), to increase cell and nucleus penetration (TAT) and to enhance endosomal escape (HisTag), besides the Rp3 to assist in the cytosol trafficking, thus covering most of the major obstacles to the vectors in intracellular level. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. Gel retardation assays and Malvern Zetasizer studies indicate an efficient complex formation between pDNA and the fusion protein. Transfection of cultured HeLa cells indicate a much higher transfection efficiency when compared to the positive control Protamine and to RD4 (the same construction but without TAT). For instance, pDNA:TAT-RD4 in the molar ratio 1:8000 was over 900 times more efficient than particles formed by the nuclear protein Protamine at the same pDNA:protein molar ratio. This study is part of an effort to develop modular shuttle proteins that successfully mimic the effects of viral vectors, in order to develop vectors for gene delivery that are both safe and efficient.
637. Effect of PEG Grafting Degree on Morphology and Transfection Efficiency of Polycation-g-PEG/DNA Nanoparticles
John-Michael Williford,1,2,3 Yong Ren,1 Xuan Jiang,1 Hai-Quan Mao.1,2,3 1 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD; 2Translational Tissue Engineering Center, Johns Hopkins University, Baltimore, MD; 3Department of Biomedical Engineering, Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, MD.
Despite the wide range of polymer/DNA nanoparticles developed for gene therapy applications, there has been no systematic study to understand the role of nanoparticle shape on their transfection efficiency and transport properties. This is primarily due to the lack of a suitable method to control the shape of DNA-containing nanoparticles. Using a self-assembly method to prepare nanoparticles from DNA and poly(ethylene glycol) (PEG)-polycation copolymers, we have been able to control the shape of the polymer/DNA nanoparticles by altering the polymer structure and assembly conditions. In this study, we analyzed the effects of PEG grafting degree on nanoparticle shape and subsequent in vitro and in vivo transfection efficiency using two different polycations-linear polyethylenimine (LPEI) and polyphosphoramidate (PPA). LPEIg-PEG/DNA nanoparticles prepared with low PEG grafting degrees Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
assumed compact spherical and short rod morphology, whereas those prepared with high PEG grafting degrees form longer rod and worm-like structures. PPA-g-PEG/DNA nanoparticles prepared with low grafting degree also form compact spherical and short rod morphologies, and particles prepared with a higher grafted polymer form predominantly long worm morphologies (Fig. 1). Furthermore, grafting of PEG to LPEI using a disulfide bond allowed for shape conversion from worm-like to short rod and spherical nanoparticles upon cleavage of a fraction of the PEG chains under reducing conditions. In vitro transfection efficiency in HEK293 cells demonstrated shape-dependent transfection efficiencies. Spherical and short-rod PPA-g-PEG/DNA nanoparticles mediated 40-fold higher efficiency compared to worm-shaped nanoparticles (Fig. 1). In vivo transfection of PPA-g-PEG/DNA nanoparticles was also analyzed using in vivo bioluminescence imaging following retrograde intrabiliary infusion of nanoparticles. Results showed that wormlike nanoparticles achieve 1.5-fold higher transfection efficiency in rat liver compared spherical and short-rod particles 24 hours after administration. Using this system, we can effectively control the shape of polymer/DNA nanoparticles using PEG-polycation graft copolymers and can further investigate the role of nanoparticle shape on transfection and transport in biological systems.
Figure 1: TEM images of (a) LPEI9K-g-PEG5K(0.5%)/DNA, (b) LPEI9K-g-PEG5K(4%)/DNA, (c) PPA31K-g-PEG5K(0.5%)/DNA, and (d) PPA31K-g-PEG10K(4%)/DNA nanoparticles. Numbers in parenthesis denote mol% of amine groups grafted with PEG.
638. Combinational Anti-Cancer Therapy with Doxorubicin and EGFR-Targeted Immunonanoparicles Containing IL12/Salmosin Genes
Jung Seok Kim,1 Yeon Kyung Lee,1 Hwa Yeon Jeong,1 Young Eun Shin,1 Seong Jae Kang,1 Min Woo Kim,1 Hong Sung Kim,2 Keun Sik Kim,3 Yong Serk Park.1 1 Biomedical Laboratory Science, Yonsei University, Wonju, Republic of Korea; 2Biomedical Laboratory Science, Korea Nazarene University, Cheonan, Republic of Korea; 3Biosciences and Biotechnology, Konyang University, Daejeon, Republic of Korea. A variety of liposome-based gene delivery systems combining targeting and treatment abilities have been developed for applications in cancer therapy. Among the liposomal vectors, immunoliposomal formulas have been suggested as a next generation of targeted delivery system. Previous systematic in vitro and in vivo studies have shown that targeting of epidermal growth factor receptor S245
Marianna T. P. Favaro,1 Marcelo A. S. de Toledo,1 Rafael F. Alves,2 Lucimara G. de la Torre,3 Anete P. Souza,1 Adriano R. Azzoni.2 1 Molecular Biology and Genetic Engineering Center, State University of Campinas, Campinas, Brazil; 2Chemical Engineering Department, University of São Paulo, Brazil; 3Faculty of Chemical Engineering, State University of Campinas, Campinas, Brazil. One of the major challenges faced by gene therapy and DNA vaccination is the development of a delivery vector that is both efficient and safe. Although much more effective, viral vectors still raise several concerns about its safety. In this context “artificial viruses” are interesting options, since they are non-viral vectors intended to explore the cell’s architecture in an efficient way, to overcome a series of physical, enzymatic and diffusional barriers, while still preserving the safety of plasmid DNA (pDNA) vectors. The main objective of this work is to exploit molecular motors, like dynein, to transport cargoes from the periphery to the centrosome of mammalian cells via the microtubule network. For that, human dynein light chain Rp3 was fusioned to a N-terminal DNA binding domain and a C-terminal membrane active peptide, TAT. The protein, named TAT-RD4, has additionally a HisTag. The shuttle protein built contains therefore different domains to promote pDNA condensation (DNA binding), to increase cell and nucleus penetration (TAT) and to enhance endosomal escape (HisTag), besides the Rp3 to assist in the cytosol trafficking, thus covering most of the major obstacles to the vectors in intracellular level. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. Gel retardation assays and Malvern Zetasizer studies indicate an efficient complex formation between pDNA and the fusion protein. Transfection of cultured HeLa cells indicate a much higher transfection efficiency when compared to the positive control Protamine and to RD4 (the same construction but without TAT). For instance, pDNA:TAT-RD4 in the molar ratio 1:8000 was over 900 times more efficient than particles formed by the nuclear protein Protamine at the same pDNA:protein molar ratio. This study is part of an effort to develop modular shuttle proteins that successfully mimic the effects of viral vectors, in order to develop vectors for gene delivery that are both safe and efficient.
637. Effect of PEG Grafting Degree on Morphology and Transfection Efficiency of Polycation-g-PEG/DNA Nanoparticles
John-Michael Williford,1,2,3 Yong Ren,1 Xuan Jiang,1 Hai-Quan Mao.1,2,3 1 Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD; 2Translational Tissue Engineering Center, Johns Hopkins University, Baltimore, MD; 3Department of Biomedical Engineering, Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, MD.
Despite the wide range of polymer/DNA nanoparticles developed for gene therapy applications, there has been no systematic study to understand the role of nanoparticle shape on their transfection efficiency and transport properties. This is primarily due to the lack of a suitable method to control the shape of DNA-containing nanoparticles. Using a self-assembly method to prepare nanoparticles from DNA and poly(ethylene glycol) (PEG)-polycation copolymers, we have been able to control the shape of the polymer/DNA nanoparticles by altering the polymer structure and assembly conditions. In this study, we analyzed the effects of PEG grafting degree on nanoparticle shape and subsequent in vitro and in vivo transfection efficiency using two different polycations-linear polyethylenimine (LPEI) and polyphosphoramidate (PPA). LPEIg-PEG/DNA nanoparticles prepared with low PEG grafting degrees Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
assumed compact spherical and short rod morphology, whereas those prepared with high PEG grafting degrees form longer rod and worm-like structures. PPA-g-PEG/DNA nanoparticles prepared with low grafting degree also form compact spherical and short rod morphologies, and particles prepared with a higher grafted polymer form predominantly long worm morphologies (Fig. 1). Furthermore, grafting of PEG to LPEI using a disulfide bond allowed for shape conversion from worm-like to short rod and spherical nanoparticles upon cleavage of a fraction of the PEG chains under reducing conditions. In vitro transfection efficiency in HEK293 cells demonstrated shape-dependent transfection efficiencies. Spherical and short-rod PPA-g-PEG/DNA nanoparticles mediated 40-fold higher efficiency compared to worm-shaped nanoparticles (Fig. 1). In vivo transfection of PPA-g-PEG/DNA nanoparticles was also analyzed using in vivo bioluminescence imaging following retrograde intrabiliary infusion of nanoparticles. Results showed that wormlike nanoparticles achieve 1.5-fold higher transfection efficiency in rat liver compared spherical and short-rod particles 24 hours after administration. Using this system, we can effectively control the shape of polymer/DNA nanoparticles using PEG-polycation graft copolymers and can further investigate the role of nanoparticle shape on transfection and transport in biological systems.
Figure 1: TEM images of (a) LPEI9K-g-PEG5K(0.5%)/DNA, (b) LPEI9K-g-PEG5K(4%)/DNA, (c) PPA31K-g-PEG5K(0.5%)/DNA, and (d) PPA31K-g-PEG10K(4%)/DNA nanoparticles. Numbers in parenthesis denote mol% of amine groups grafted with PEG.
638. Combinational Anti-Cancer Therapy with Doxorubicin and EGFR-Targeted Immunonanoparicles Containing IL12/Salmosin Genes
Jung Seok Kim,1 Yeon Kyung Lee,1 Hwa Yeon Jeong,1 Young Eun Shin,1 Seong Jae Kang,1 Min Woo Kim,1 Hong Sung Kim,2 Keun Sik Kim,3 Yong Serk Park.1 1 Biomedical Laboratory Science, Yonsei University, Wonju, Republic of Korea; 2Biomedical Laboratory Science, Korea Nazarene University, Cheonan, Republic of Korea; 3Biosciences and Biotechnology, Konyang University, Daejeon, Republic of Korea. A variety of liposome-based gene delivery systems combining targeting and treatment abilities have been developed for applications in cancer therapy. Among the liposomal vectors, immunoliposomal formulas have been suggested as a next generation of targeted delivery system. Previous systematic in vitro and in vivo studies have shown that targeting of epidermal growth factor receptor S245