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168. Efficient Non-Viral, Large-Scale, Gene Transfer for Ex Vivo Cell Transdifferentiation
NAKED DNA: METHODS 167. Atelocollagen-Mediated Delivery System for Long-Acting Nucleic Acid Medicinal Therapies
168. Efficient Non-Viral, Large-Scale, Gene Transfer for Ex Vivo Cell Transdifferentiation
Shunji Nagahara,1 Takahiro Ochiya,2 Akihiko Sano,1 Hiroshi Ito,3 Kimi Honma,3 Masaaki Terada.2 1 Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan; 2National Cancer Research Institute, Tokyo, Japan; 3Koken Co., Ltd., Tokyo, Japan.
Linda N. Liu,1 Lin-Hong Li,1 Cornell Allen,1 Rama Shivakumar,1 Stephanie Feller,1 Jonathan M. Weiss,1 Joseph C. Fratantoni.1 1 Gene Delivery, MaxCyte, Inc., Rockville, MD, United States.
Much effort has been spent on the development of a method of introducing nucleic acid medicines (NAMs), such as plasmid DNA (pDNA) for gene therapy or DNA vaccination and oligonucleotides for antisense therapy, into cells. The progress of delivery technology using liposomes and cationic lipids made it possible to introduce these NAMs into cells efficiently. However, the period within which NAMs act effectively has been hardly investigated. Indeed, gene expression using pDNA and inhibition of gene expression using oligonucleotides are transient. The transiency is an obstacle to the progress of therapies using NAMs for practical use. In this study, we demonstrate the properties of an atelocollagen-mediated delivery system, in which we developed for long-acting nucleic acid medicinal therapies. Atelocollagen is obtained by pepsin digestion of collagen to remove telopeptides in which most of the antigenicity is included, and is widely used because of its high biocompatibility derived from its low toxicity and biodegradability. Prolongation of therapeutic effects of NAMs was investigated with three types of formulations as follows. The Minipellet is a cylindrical formulation, in which pDNA is encapsulated with the atelocollagen matrix. When Minipellet (10mm in length and 0.6mm in diameter) containing a pDNA encoding HST-1, which is an inducer of blood platelet, was injected intramuscularly into mice, the release of pDNA to the peripheral blood persisted over a period of 40 days. The serum HST-1 concentration and the blood platelet count increased gradually following the injection and remained at a high level for at least 60 days. In contrast, injection of pDNA solution resulted in only a transient increase in the serum HST-1 cncentration and blood plateletcount. These results indicate that injection of the Minipellet containing pDNA results in the production of physiologically useful levels of encoded-protein, which is maintained over a long period. pDNA forms a complex with atelocollagen, a pDNA/atelocollagen complex, by electrostatic interaction, and the complex can be injected into the living body. The complex containing a pDNA encoding the envelope protein of the hepatitis B virus was prepared. Subcutaneous injection of the complex induced a relatively sustainable high level of specific immune responses compared with the pDNA injection. This result indicates that the complex is able to enhance immune responses and extends the period of immunization by DNA vaccination. In the same manner, an oligonucleotide forms an electrostatic complex with atelocollagen, an oligonucleotide/ atelocollagen complex. Antisense oligonucleotides forming the complex inhibit targeted gene expression for long periods compared with oligonucleotides administered with liposomes. The Minipellet and these complexes can be distributed in the injectable form, and the Minipellet containing interferon had been filed to MHW. Therefore, the atelocollagen-mediated delivery system will help in the advancement of the practical use of NAMs. Furthermore, longacting nucleic acid medicinal therapies will mitigate the patients burden by not only reducing the number of times of medication but also by reducing the medical treatment cost.
S66
Forced expression of certain transcription-regulated factors can guide adult cells and stem cells into other lineages and has great potential for cell-based therapeutics and tissue regeneration. A transient gene-transfer (GT) system with capacities to process large amount of cells will benefit the field fundamentally. Electroporation (EP) is used to transfect and load cells with various molecules, such as DNA. However, traditional EP using a static mode is typically restricted to volumes less than 1 mL, limiting its use in clinical and industrial settings. Here we report results from an efficient, fully scalable, EP-based GT system and demonstrate its suitability for ex vivo cell transdifferentiation. We used the well-characterized muscle-specific factor, MyoD, to test the feasibility of MaxCyte GT for transdifferentiating primary mouse fibroblasts (10T1/2) into myoblasts. 10T1/2 cells were first transfected in a small volume setting (5e6 cells in 100 μL) with a DNA plasmid coding for GFP. Greater than 90% of the cells expressed GFP when analyzed by FACS 48 hr post transfection. Propidium iodide staining exclusion revealed that greater than 90% of the cells were viable. Full-length cDNA encoding human MyoD was subcloned into the same site on the pCI backbone as the GFP construct and then electroporated into 10T1/2 cells using the pulsing parameters obtained by GFP optimization. The transfected cells were plated in growth media and allowed to attach overnight. Then the cells were switched to and maintained in differentiation media. By day 5, the cells had started to fuse and form multinucleated cells. Immunohistochemical staining of the cells at 10 days post transfection confirmed that greater than 60% of the monolayer now expressed myosin heavy chain, a marker for myogenic differentiation. These data demonstrate that MaxCyte GT system can efficiently deliver transgenes to primary cells and promote transdifferentiation. The process may be scaled up to a level more suitable for clinic application. Suspension human lymphocytic Jurkat cells, which can be prepared easily in large number, were used as target cells to develop the flow EP-based GT system. 2.5 billion Jurkat cells were resuspended in 50 mLs of EP buffer with DNA plasmid coding for eGFP. The cell/DNA mixture was circulated and processed into a chamber comprising of a pair of electrodes, which were 5 mm apart, at a speed of 6 mL/min. The processed cells were collected every 10 mLs during EP and cultured 48 hours prior to FACS analysis. Approximately 75% of the cells expressed GFP, and greater than 80% of the cells were viable. Most importantly, the transgene expression and cell viability results were identical among samples collected at different time points during processing, suggesting that the system can be scaled up to volumes greater than 50 mLs. The results were also close to those obtained from small volume settings. Currently, large-scale transdifferentiation is under investigation. In conclusion, the MaxCyte non-viral GT system can handle rapid cell loading including gene transfer in large-scale, which is most suitable for clinically relevant volumes for ex vivo cell transdifferentiation and regenerative medicine.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
168. Efficient Non-Viral, Large-Scale, Gene Transfer for Ex Vivo Cell Transdifferentiation
Shunji Nagahara,1 Takahiro Ochiya,2 Akihiko Sano,1 Hiroshi Ito,3 Kimi Honma,3 Masaaki Terada.2 1 Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan; 2National Cancer Research Institute, Tokyo, Japan; 3Koken Co., Ltd., Tokyo, Japan.
Linda N. Liu,1 Lin-Hong Li,1 Cornell Allen,1 Rama Shivakumar,1 Stephanie Feller,1 Jonathan M. Weiss,1 Joseph C. Fratantoni.1 1 Gene Delivery, MaxCyte, Inc., Rockville, MD, United States.
Much effort has been spent on the development of a method of introducing nucleic acid medicines (NAMs), such as plasmid DNA (pDNA) for gene therapy or DNA vaccination and oligonucleotides for antisense therapy, into cells. The progress of delivery technology using liposomes and cationic lipids made it possible to introduce these NAMs into cells efficiently. However, the period within which NAMs act effectively has been hardly investigated. Indeed, gene expression using pDNA and inhibition of gene expression using oligonucleotides are transient. The transiency is an obstacle to the progress of therapies using NAMs for practical use. In this study, we demonstrate the properties of an atelocollagen-mediated delivery system, in which we developed for long-acting nucleic acid medicinal therapies. Atelocollagen is obtained by pepsin digestion of collagen to remove telopeptides in which most of the antigenicity is included, and is widely used because of its high biocompatibility derived from its low toxicity and biodegradability. Prolongation of therapeutic effects of NAMs was investigated with three types of formulations as follows. The Minipellet is a cylindrical formulation, in which pDNA is encapsulated with the atelocollagen matrix. When Minipellet (10mm in length and 0.6mm in diameter) containing a pDNA encoding HST-1, which is an inducer of blood platelet, was injected intramuscularly into mice, the release of pDNA to the peripheral blood persisted over a period of 40 days. The serum HST-1 concentration and the blood platelet count increased gradually following the injection and remained at a high level for at least 60 days. In contrast, injection of pDNA solution resulted in only a transient increase in the serum HST-1 cncentration and blood plateletcount. These results indicate that injection of the Minipellet containing pDNA results in the production of physiologically useful levels of encoded-protein, which is maintained over a long period. pDNA forms a complex with atelocollagen, a pDNA/atelocollagen complex, by electrostatic interaction, and the complex can be injected into the living body. The complex containing a pDNA encoding the envelope protein of the hepatitis B virus was prepared. Subcutaneous injection of the complex induced a relatively sustainable high level of specific immune responses compared with the pDNA injection. This result indicates that the complex is able to enhance immune responses and extends the period of immunization by DNA vaccination. In the same manner, an oligonucleotide forms an electrostatic complex with atelocollagen, an oligonucleotide/ atelocollagen complex. Antisense oligonucleotides forming the complex inhibit targeted gene expression for long periods compared with oligonucleotides administered with liposomes. The Minipellet and these complexes can be distributed in the injectable form, and the Minipellet containing interferon had been filed to MHW. Therefore, the atelocollagen-mediated delivery system will help in the advancement of the practical use of NAMs. Furthermore, longacting nucleic acid medicinal therapies will mitigate the patients burden by not only reducing the number of times of medication but also by reducing the medical treatment cost.
S66
Forced expression of certain transcription-regulated factors can guide adult cells and stem cells into other lineages and has great potential for cell-based therapeutics and tissue regeneration. A transient gene-transfer (GT) system with capacities to process large amount of cells will benefit the field fundamentally. Electroporation (EP) is used to transfect and load cells with various molecules, such as DNA. However, traditional EP using a static mode is typically restricted to volumes less than 1 mL, limiting its use in clinical and industrial settings. Here we report results from an efficient, fully scalable, EP-based GT system and demonstrate its suitability for ex vivo cell transdifferentiation. We used the well-characterized muscle-specific factor, MyoD, to test the feasibility of MaxCyte GT for transdifferentiating primary mouse fibroblasts (10T1/2) into myoblasts. 10T1/2 cells were first transfected in a small volume setting (5e6 cells in 100 μL) with a DNA plasmid coding for GFP. Greater than 90% of the cells expressed GFP when analyzed by FACS 48 hr post transfection. Propidium iodide staining exclusion revealed that greater than 90% of the cells were viable. Full-length cDNA encoding human MyoD was subcloned into the same site on the pCI backbone as the GFP construct and then electroporated into 10T1/2 cells using the pulsing parameters obtained by GFP optimization. The transfected cells were plated in growth media and allowed to attach overnight. Then the cells were switched to and maintained in differentiation media. By day 5, the cells had started to fuse and form multinucleated cells. Immunohistochemical staining of the cells at 10 days post transfection confirmed that greater than 60% of the monolayer now expressed myosin heavy chain, a marker for myogenic differentiation. These data demonstrate that MaxCyte GT system can efficiently deliver transgenes to primary cells and promote transdifferentiation. The process may be scaled up to a level more suitable for clinic application. Suspension human lymphocytic Jurkat cells, which can be prepared easily in large number, were used as target cells to develop the flow EP-based GT system. 2.5 billion Jurkat cells were resuspended in 50 mLs of EP buffer with DNA plasmid coding for eGFP. The cell/DNA mixture was circulated and processed into a chamber comprising of a pair of electrodes, which were 5 mm apart, at a speed of 6 mL/min. The processed cells were collected every 10 mLs during EP and cultured 48 hours prior to FACS analysis. Approximately 75% of the cells expressed GFP, and greater than 80% of the cells were viable. Most importantly, the transgene expression and cell viability results were identical among samples collected at different time points during processing, suggesting that the system can be scaled up to volumes greater than 50 mLs. The results were also close to those obtained from small volume settings. Currently, large-scale transdifferentiation is under investigation. In conclusion, the MaxCyte non-viral GT system can handle rapid cell loading including gene transfer in large-scale, which is most suitable for clinically relevant volumes for ex vivo cell transdifferentiation and regenerative medicine.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy