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Evans MM, Liu S, Krautner JS, Seguin CG, Leung R, Ronald JA. Evaluation of DNA minicircles for delivery of adenine and cytosine base editors using activatable gene on "GO" reporter imaging systems. MOLECULAR THERAPY. NUCLEIC ACIDS 2024; 35:102248. [PMID: 39040503 PMCID: PMC11260848 DOI: 10.1016/j.omtn.2024.102248] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/11/2023] [Accepted: 06/07/2024] [Indexed: 07/24/2024]
Abstract
Over 30,000 point mutations are associated with debilitating diseases, including many cancer types, underscoring a critical need for targeted genomic solutions. CRISPR base editors, like adenine base editors (ABEs) and cytosine base editors (CBEs), enable precise modifications by converting adenine to guanine and cytosine to thymine, respectively. Challenges in efficiency and safety concerns regarding viral vectors used in delivery limit the scope of base editing. This study introduces non-viral minicircles, bacterial-backbone-free plasmids, as a delivery vehicle for ABEs and CBEs. The research uses cells engineered with the "Gene On" (GO) reporter gene systems for tracking minicircle-delivered ABEs, CBEs, or Cas9 nickase (control), using green fluorescent protein (GFPGO), bioluminescence reporter firefly luciferase (LUCGO), or a highly sensitive Akaluciferase (AkalucGO) designed in this study. The results show that transfection of minicircles expressing CBE or ABE resulted in significantly higher GFP expression and luminescence signals over controls, with minicircles demonstrating the most substantial editing. This study presents minicircles as a new strategy for base editor delivery and develops an enhanced bioluminescence imaging reporter system for tracking ABE activity. Future studies aim to evaluate the use of minicircles in preclinical cancer models, facilitating potential clinical applications.
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Affiliation(s)
- Melissa M. Evans
- Robarts Research Institute, University of Western Ontario, London, ON N6A 3K7, Canada
- Department of Medical Biophysics, University of Western Ontario, London, ON N6A 5C1, Canada
| | - Shirley Liu
- Robarts Research Institute, University of Western Ontario, London, ON N6A 3K7, Canada
- Department of Medical Biophysics, University of Western Ontario, London, ON N6A 5C1, Canada
| | - Joshua S. Krautner
- Robarts Research Institute, University of Western Ontario, London, ON N6A 3K7, Canada
- Department of Medical Biophysics, University of Western Ontario, London, ON N6A 5C1, Canada
| | - Caroline G. Seguin
- Robarts Research Institute, University of Western Ontario, London, ON N6A 3K7, Canada
| | - Rajan Leung
- Robarts Research Institute, University of Western Ontario, London, ON N6A 3K7, Canada
- Department of Medical Biophysics, University of Western Ontario, London, ON N6A 5C1, Canada
| | - John A. Ronald
- Robarts Research Institute, University of Western Ontario, London, ON N6A 3K7, Canada
- Department of Medical Biophysics, University of Western Ontario, London, ON N6A 5C1, Canada
- Lawson Health Research Institute, London, ON N6C 2R5, Canada
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2
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Eom YJ, Kim JW, Rim YA, Lim J, Jung SI, Ju JH. Effects of stepwise administration of osteoprotegerin and parathyroid hormone-related peptide DNA vectors on bone formation in ovariectomized rat model. Sci Rep 2024; 14:2477. [PMID: 38291053 PMCID: PMC10827729 DOI: 10.1038/s41598-024-51957-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2023] [Accepted: 01/11/2024] [Indexed: 02/01/2024] Open
Abstract
Osteoporosis is a metabolic bone disease that impairs bone mineral density, microarchitecture, and strength. It requires continuous management, and further research into new treatment options is necessary. Osteoprotegerin (OPG) inhibits bone resorption and osteoclast activity. The objective of this study was to investigate the effects of stepwise administration of OPG-encoded minicircles (mcOPG) and a bone formation regulator, parathyroid hormone-related peptide (PTHrP)-encoded minicircles (mcPTHrP) in osteoporosis. The combined treatment with mcOPG and mcPTHrP significantly increased osteogenic marker expression in osteoblast differentiation compared with the single treatment groups. A model of postmenopausal osteoporosis was established in 12-week-old female rats through ovariectomy (OVX). After 8 weeks of OVX, mcOPG (80 µg/kg) was administered via intravenous injection. After 16 weeks of OVX, mcPTHrP (80 µg/kg) was injected once a week for 3 weeks. The bone microstructure in the femur was evaluated 24 weeks after OVX using micro-CT. In a proof-of-concept study, stepwise treatment with mcOPG and mcPTHrP on an OVX rat model significantly improved bone microstructure compared to treatment with mcOPG or mcPTHrP alone. These results suggest that stepwise treatment with mcOPG and mcPTHrP may be a potential treatment for osteoporosis.
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Affiliation(s)
- Ye Ji Eom
- Catholic iPSC Research Center (CiRC), CiSTEM Laboratory, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
- Department of Biomedicine and Health Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Jang-Woon Kim
- Catholic iPSC Research Center (CiRC), CiSTEM Laboratory, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Yeri Alice Rim
- Catholic iPSC Research Center (CiRC), CiSTEM Laboratory, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
| | - Jooyoung Lim
- Catholic iPSC Research Center (CiRC), CiSTEM Laboratory, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
- Department of Biomedicine and Health Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Se In Jung
- Catholic iPSC Research Center (CiRC), CiSTEM Laboratory, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
- Department of Biomedicine and Health Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
| | - Ji Hyeon Ju
- Catholic iPSC Research Center (CiRC), CiSTEM Laboratory, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
- Division of Rheumatology, Department of Internal Medicine, Institute of Medical Science, College of Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, Seoul, Republic of Korea.
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3
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Safdar M, Ullah M, Wahab A, Hamayun S, Ur Rehman M, Khan MA, Khan SU, Ullah A, Din FU, Awan UA, Naeem M. Genomic insights into heart health: Exploring the genetic basis of cardiovascular disease. Curr Probl Cardiol 2024; 49:102182. [PMID: 37913933 DOI: 10.1016/j.cpcardiol.2023.102182] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Accepted: 10/28/2023] [Indexed: 11/03/2023]
Abstract
Cardiovascular diseases (CVDs) are considered as the leading cause of death worldwide. CVD continues to be a major cause of death and morbidity despite significant improvements in its detection and treatment. Therefore, it is strategically important to be able to precisely characterize an individual's sensitivity to certain illnesses. The discovery of genes linked to cardiovascular illnesses has benefited from linkage analysis and genome-wide association research. The last 20 years have seen significant advancements in the field of molecular genetics, particularly with the development of new tools like genome-wide association studies. In this article we explore the profound impact of genetic variations on disease development, prognosis, and therapeutic responses. And the significance of genetics in cardiovascular risk assessment and the ever-evolving realm of genetic testing, offering insights into the potential for personalized medicine in this domain. Embracing the future of cardiovascular care, the article explores the implications of pharmacogenomics for tailored treatments, the promise of emerging technologies in cardiovascular genetics and therapies, including the transformative influence of nanotechnology. Furthermore, it delves into the exciting frontiers of gene editing, such as CRISPR/Cas9, as a novel approach to combat cardiovascular diseases. And also explore the potential of stem cell therapy and regenerative medicine, providing a holistic view of the dynamic landscape of cardiovascular genomics and its transformative potential for the field of cardiovascular medicine.
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Affiliation(s)
- Mishal Safdar
- Department of Biological Sciences, National University of Medical Sciences (NUMS) Rawalpindi 46000, Punjab, Pakistan
| | - Muneeb Ullah
- Department of Pharmacy, Kohat University of Science, and technology (KUST), Kohat, 26000, Khyber Pakhtunkhwa, Pakistan
| | - Abdul Wahab
- Department of Pharmacy, Kohat University of Science, and technology (KUST), Kohat, 26000, Khyber Pakhtunkhwa, Pakistan
| | - Shah Hamayun
- Department of Cardiology, Pakistan Institute of Medical Sciences (PIMS), Islamabad, 04485 Punjab, Pakistan
| | - Mahboob Ur Rehman
- Department of Cardiology, Pakistan Institute of Medical Sciences (PIMS), Islamabad, 04485 Punjab, Pakistan
| | - Muhammad Amir Khan
- Department of Foreign Medical education, Fergana Medical institute of Public Health, 2A Yangi Turon street, Fergana 150100, Uzbekistan
| | - Shahid Ullah Khan
- Department of Biochemistry, Women Medical and Dental College, Khyber Medical University, Abbottabad, 22080, Khyber Pakhtunkhwa, Pakistan
| | - Aziz Ullah
- Department of Chemical Engineering, Pukyong National University, Busan 48513, Republic of Korea
| | - Fakhar Ud Din
- Department of Pharmacy, Quaid-i-Azam University, 45320, Islamabad, Pakistan
| | - Uzma Azeem Awan
- Department of Biological Sciences, National University of Medical Sciences (NUMS) Rawalpindi 46000, Punjab, Pakistan
| | - Muhammad Naeem
- Department of Biological Sciences, National University of Medical Sciences (NUMS) Rawalpindi 46000, Punjab, Pakistan.
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Liu CM, Chen YC, Hu YF. Harnessing cell reprogramming for cardiac biological pacing. J Biomed Sci 2023; 30:74. [PMID: 37633890 PMCID: PMC10463311 DOI: 10.1186/s12929-023-00970-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2023] [Accepted: 08/22/2023] [Indexed: 08/28/2023] Open
Abstract
Electrical impulses from cardiac pacemaker cardiomyocytes initiate cardiac contraction and blood pumping and maintain life. Abnormal electrical impulses bring patients with low heart rates to cardiac arrest. The current therapy is to implant electronic devices to generate backup electricity. However, complications inherent to electronic devices remain unbearable suffering. Therefore, cardiac biological pacing has been developed as a hardware-free alternative. The approaches to generating biological pacing have evolved recently using cell reprogramming technology to generate pacemaker cardiomyocytes in-vivo or in-vitro. Different from conventional methods by electrical re-engineering, reprogramming-based biological pacing recapitulates various phenotypes of de novo pacemaker cardiomyocytes and is more physiological, efficient, and easy for clinical implementation. This article reviews the present state of the art in reprogramming-based biological pacing. We begin with the rationale for this new approach and review its advances in creating a biological pacemaker to treat bradyarrhythmia.
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Affiliation(s)
- Chih-Min Liu
- Division of Cardiology, Department of Medicine, Heart Rhythm Center, Taipei Veterans General Hospital, Taipei, Taiwan
- Faculty of Medicine and Institute of Clinical Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yi-Chun Chen
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
- Institute of Biopharmaceutical Sciences, College of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yu-Feng Hu
- Division of Cardiology, Department of Medicine, Heart Rhythm Center, Taipei Veterans General Hospital, Taipei, Taiwan.
- Faculty of Medicine and Institute of Clinical Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan.
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
- Institute of Biopharmaceutical Sciences, College of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan.
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Staurenghi F, McClements ME, Salman A, MacLaren RE. Minicircle Delivery to the Neural Retina as a Gene Therapy Approach. Int J Mol Sci 2022; 23:11673. [PMID: 36232975 PMCID: PMC9569440 DOI: 10.3390/ijms231911673] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 09/26/2022] [Accepted: 09/28/2022] [Indexed: 11/07/2022] Open
Abstract
Non-viral gene therapy has the potential to overcome several shortcomings in viral vector-based therapeutics. Methods of in vivo plasmid delivery have developed over recent years to increase the efficiency of non-viral gene transfer, yet further improvements still need to be made to improve their translational capacity. Gene therapy advances for inherited retinal disease have been particularly prominent over the recent decade but overcoming physical and physiological barriers present in the eye remains a key obstacle in the field of non-viral ocular drug delivery. Minicircles are circular double-stranded DNA vectors that contain expression cassettes devoid of bacterial DNA, thereby limiting the risks of innate immune responses induced by such elements. To date, they have not been extensively used in pre-clinical studies yet remain a viable vector option for the treatment of inherited retinal disease. Here, we explore the potential of minicircle DNA delivery to the neural retina as a gene therapy approach. We consider the advantages of minicircles as gene therapy vectors as well as review the challenges involved in optimising their delivery to the neural retina.
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Affiliation(s)
- Federica Staurenghi
- Nuffield Laboratory of Ophthalmology, Clinical Neurosciences, University of Oxford, Oxford OX3 9DU, UK
| | - Michelle E. McClements
- Nuffield Laboratory of Ophthalmology, Clinical Neurosciences, University of Oxford, Oxford OX3 9DU, UK
| | - Ahmed Salman
- Nuffield Laboratory of Ophthalmology, Clinical Neurosciences, University of Oxford, Oxford OX3 9DU, UK
| | - Robert E. MacLaren
- Nuffield Laboratory of Ophthalmology, Clinical Neurosciences, University of Oxford, Oxford OX3 9DU, UK
- Oxford University Hospital, Oxford OX3 9DU, UK
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Lim SW, Shin YJ, Cui S, Ko EJ, Yoo SH, Chung BH, Yang CW. Therapeutic effect of multiple functional minicircle vector encoding anti-CD25/IL-10/CXCR3 in allograft rejection model. Korean J Intern Med 2022; 37:1031-1049. [PMID: 35725307 PMCID: PMC9449213 DOI: 10.3904/kjim.2021.299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Accepted: 01/04/2022] [Indexed: 12/03/2022] Open
Abstract
BACKGROUND/AIMS We previously proposed minicircle vector technology as the potential platform for the development and production of new biologics. In this study, we have designed a novel target molecule for the treatment of allograft rejection and evaluated its feasibility as the therapeutic agent in this disease using the minicircle vector system. METHODS We engineered vectors to carry cassette sequences for anti-CD25, interleukin-10 (IL-10), and C-X-C motif chemokine receptor 3 (CXCR3) fusion protein, and then isolated minicircle vectors from the parent vectors. We verified the substantial production of anti-CD25/IL-10/CXCR3 fusion protein from minicircles and their duration in HEK293T cells and mice models. We also evaluated whether minicircle-derived anti-CD25/IL-10/CXCR3 has therapeutic effects in a skin allograft in mice model. RESULTS We confirmed the production of anti-CD25/IL-10/CXCR3 from minicircle by its significant availability in cells transfected with the minicircle and in its conditioned media. After a single injection of minicircle by hydrodynamic injection via mouse tail vein, luminescence or red fluorescence was maintained until 40 days in the liver tissue, suggesting the production of anti-CD25/IL-10/CXCR3 protein from minicircles via protein synthesis machinery in the liver. Mice treated with the minicircle encoding anti-CD25/IL-10/CXCR3 showed prolonged skin allograft survival times accompanied by improved immunologic regulation e.g., reduction of the lymphocyte population of Th1, Th2, and Th17 and an induction of regulatory T cells. CONCLUSION These findings implied that self-generated anti-CD25/IL-10/CXCR3 protein drug by minicircle technology is functionally active and relevant for reducing allograft rejection. The minicircle vector system may be useful for developing new biological drugs, avoiding manufacturing or practical problems.
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Affiliation(s)
- Sun Woo Lim
- Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
| | - Yoo Jin Shin
- Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
| | - Sheng Cui
- Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
| | - Eun Jeong Ko
- Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Division of Nephrology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
| | | | - Byung Ha Chung
- Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Division of Nephrology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
| | - Chul Woo Yang
- Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
- Division of Nephrology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul,
Korea
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7
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Gara E, Ong SG, Winkler J, Zlabinger K, Lukovic D, Merkely B, Emmert MY, Wolint P, Hoerstrup SP, Gyöngyösi M, Wu JC, Pavo N. Cell-Based HIF1α Gene Therapy Reduces Myocardial Scar and Enhances Angiopoietic Proteome, Transcriptomic and miRNA Expression in Experimental Chronic Left Ventricular Dysfunction. Front Bioeng Biotechnol 2022; 10:767985. [PMID: 35646882 PMCID: PMC9133350 DOI: 10.3389/fbioe.2022.767985] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Accepted: 04/14/2022] [Indexed: 11/13/2022] Open
Abstract
Recent preclinical investigations and clinical trials with stem cells mostly studied bone-marrow-derived mononuclear cells (BM-MNCs), which so far failed to meet clinically significant functional study endpoints. BM-MNCs containing small proportions of stem cells provide little regenerative potential, while mesenchymal stem cells (MSCs) promise effective therapy via paracrine impact. Genetic engineering for rationally enhancing paracrine effects of implanted stem cells is an attractive option for further development of therapeutic cardiac repair strategies. Non-viral, efficient transfection methods promise improved clinical translation, longevity and a high level of gene delivery. Hypoxia-induced factor 1α is responsible for pro-angiogenic, anti-apoptotic and anti-remodeling mechanisms. Here we aimed to apply a cellular gene therapy model in chronic ischemic heart failure in pigs. A non-viral circular minicircle DNA vector (MiCi) was used for in vitro transfection of porcine MSCs (pMSC) with HIF1α (pMSC-MiCi-HIF-1α). pMSCs-MiCi-HIF-1α were injected endomyocardially into the border zone of an anterior myocardial infarction one month post-reperfused-infarct. Cell injection was guided via 3D-guided NOGA electro-magnetic catheter delivery system. pMSC-MiCi-HIF-1α delivery improved cardiac output and reduced myocardial scar size. Abundances of pro-angiogenic proteins were analyzed 12, 24 h and 1 month after the delivery of the regenerative substances. In a protein array, the significantly increased angiogenesis proteins were Activin A, Angiopoietin, Artemin, Endothelin-1, MCP-1; and remodeling factors ADAMTS1, FGFs, TGFb1, MMPs, and Serpins. In a qPCR analysis, increased levels of angiopeptin, CXCL12, HIF-1α and miR-132 were found 24 h after cell-based gene delivery, compared to those in untreated animals with infarction and in control animals. Expression of angiopeptin increased already 12 h after treatment, and miR-1 expression was reduced at that time point. In total, pMSC overexpressing HIF-1α showed beneficial effects for treatment of ischemic injury, mediated by stimulation of angiogenesis.
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Affiliation(s)
- Edit Gara
- Heart and Vascular Centre, Semmelweis University, Budapest, Hungary
- Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria
| | - Sang-Ging Ong
- Stanford Cardiovascular Institute, Stanford, CA, United States
| | - Johannes Winkler
- Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria
| | - Katrin Zlabinger
- Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria
| | - Dominika Lukovic
- Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria
| | - Bela Merkely
- Heart and Vascular Centre, Semmelweis University, Budapest, Hungary
| | - Maximilian Y. Emmert
- Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland
- Department of Cardiothoracic and Vascular Surgery, German Heart Center Berlin, Berlin, Germany
- Department of Cardiovascular Surgery, Charité Universitätsmedizin Berlin, Berlin, Germany
| | - Petra Wolint
- Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland
| | - Simon P. Hoerstrup
- Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland
| | - Mariann Gyöngyösi
- Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria
| | - Joseph C. Wu
- Stanford Cardiovascular Institute, Stanford, CA, United States
| | - Noemi Pavo
- Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Vienna, Austria
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Ma SP, Gao XX, Zhou GQ, Zhang HK, Yang JM, Wang WJ, Song XM, Chen HY, Lu DR. Reactivation of γ-globin expression using a minicircle DNA system to treat β-thalassemia. Gene 2022; 820:146289. [PMID: 35143940 DOI: 10.1016/j.gene.2022.146289] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2021] [Revised: 01/28/2022] [Accepted: 02/03/2022] [Indexed: 12/26/2022]
Abstract
Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for β-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of β-thalassemia. In our study, we found that the transfection efficiencies of CD34+ hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of β-thalassemia.
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Affiliation(s)
- Shuang-Ping Ma
- Institutes of Health Central Plains, Xinxiang Medical University, Xinxiang 453003, China
| | - Xu-Xia Gao
- State Key Laboratory of Genetic Engineering and MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China
| | - Guo-Qiang Zhou
- State Key Laboratory of Genetic Engineering and MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China
| | - Hao-Kun Zhang
- State Key Laboratory of Genetic Engineering and MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China
| | - Jing-Min Yang
- State Key Laboratory of Genetic Engineering and MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China
| | - Wen-Juan Wang
- Department of Hematology, the first affiliated hospital of Soochow University, Suzhou, China
| | - Xian-Min Song
- Department of Hematology, Shanghai General Hospital (affiliated to Shanghai Jiao Tong University), Shanghai, China.
| | - Hong-Yan Chen
- State Key Laboratory of Genetic Engineering and MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China.
| | - Da-Ru Lu
- State Key Laboratory of Genetic Engineering and MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China.
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9
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Pang X, Chen G, Huang P, Zhang P, Liu J, Hou X, He CY, Chen P, Xie YW, Zhao J, Chen ZY. Anticancer effects of a single intramuscular dose of a minicircle DNA vector expressing anti-CD3/CD20 in a xenograft mouse model. Mol Ther Oncolytics 2022; 24:788-798. [PMID: 35317514 PMCID: PMC8908050 DOI: 10.1016/j.omto.2022.02.014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2021] [Accepted: 02/15/2022] [Indexed: 11/25/2022] Open
Abstract
Bispecific antibodies (BsAbs) are a class of promising anticancer immunotherapies. Among them, the US Food and Drug Administration (FDA)-approved blinatumomab (BLI) is very effective in eliminating the minimum residual disease (MRD) of acute lymphoblastic leukemia (ALL), resulting in long-term remission in many individuals. However, the need for months-long intravenous delivery and high cost limit its clinical acceptance. Here we demonstrate that these problems can be solved by a BsAb expressed by one intramuscular (i.m.) dose of a minicircle DNA vector (MC). In a human B lymphoma xenograft mouse model, when microcancers became detectable in bone marrow, the mice received an i.m. dose of the MC encoding the BsAb anti-CD3/CD20 (BsAb.CD20), followed by 8 subsequent intravenous (i.v.) doses, one every other day (q2d), of human T cells to serve as effectors. The treatment resulted in persistent expression of a therapeutic level of serum BsAb.CD20 and complete regression or growth retardation of the cancers in the mice. These results suggest that the i.m. MC technology can eliminate the physical and financial burdens of i.v. delivered BLI without compromising anticancer efficacy and that cancer can be treated as easily as injecting a vaccine. This, together with other superior MC features, such as safety and affordability, suggests that the i.m. MC BsAb technology has great clinical application potential.
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Affiliation(s)
- Xiaojuan Pang
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Guochuang Chen
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Ping Huang
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
| | - Peifa Zhang
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Jie Liu
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Xiaohu Hou
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Cheng-Yi He
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Ping Chen
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Yi-Wu Xie
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Jing Zhao
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
| | - Zhi-Ying Chen
- Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
- Syno Minicircle Biotechnology Co., Ltd., Shenzhen 518055, China
- Corresponding author Zhi-Ying Chen, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 1068 Xueyuan Boulevard, Shenzhen 518055, China.
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10
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Park H, Kim D, Cho B, Byun J, Kim YS, Ahn Y, Hur J, Oh YK, Kim J. In vivo therapeutic genome editing via CRISPR/Cas9 magnetoplexes for myocardial infarction. Biomaterials 2021; 281:121327. [PMID: 34952262 DOI: 10.1016/j.biomaterials.2021.121327] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 11/17/2021] [Accepted: 12/17/2021] [Indexed: 12/26/2022]
Abstract
CRISPR/Cas9-mediated gene-editing technology has gained attention as a new therapeutic method for intractable diseases. However, the use of CRISPR/Cas9 for cardiac conditions such as myocardial infarction remains challenging due to technical and biological barriers, particularly difficulties in delivering the system and targeting genes in the heart. In the present study, we demonstrated the in vivo efficacy of the CRISPR/Cas9 magnetoplexes system for therapeutic genome editing in myocardial infarction. First, we developed CRISPR/Cas9 magnetoplexes that magnetically guided CRISPR/Cas9 system to the heart for efficient in vivo therapeutic gene targeting during heart failures. We then demonstrated that the in vivo gene targeting of miR34a via these CRISPR/Cas9 magnetoplexes in a mouse model of myocardial infarction significantly improved cardiac repair and regeneration to facilitate improvements in cardiac function. These results indicated that CRISPR/Cas9 magnetoplexes represent an effective in vivo therapeutic gene-targeting platform in the myocardial infarction of heart, and that this strategy may be applicable for the treatment of a broad range of cardiac failures.
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Affiliation(s)
- Hanseul Park
- Laboratory of Stem Cells & Cell Reprogramming, Department of Chemistry, Dongguk University, Seoul, 100715, Republic of Korea
| | - Dongyoon Kim
- College of Pharmacy, Seoul National University, 1 Kwanak-ro, Seoul, 08826, Republic of Korea
| | - Byounggook Cho
- Laboratory of Stem Cells & Cell Reprogramming, Department of Chemistry, Dongguk University, Seoul, 100715, Republic of Korea
| | - Junho Byun
- College of Pharmacy, Seoul National University, 1 Kwanak-ro, Seoul, 08826, Republic of Korea
| | - Yong Sook Kim
- Biomedical Research Institute, Chonnam National University Hospital, Gwangju, 61469, Republic of Korea
| | - Youngkeun Ahn
- Department of Cardiology, Chonnam National University Hospital, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea
| | - Jin Hur
- Department of Convergence Medicine, Pusan National University School of Medicine, Yangsan, Republic of Korea
| | - Yu-Kyoung Oh
- College of Pharmacy, Seoul National University, 1 Kwanak-ro, Seoul, 08826, Republic of Korea.
| | - Jongpil Kim
- Laboratory of Stem Cells & Cell Reprogramming, Department of Chemistry, Dongguk University, Seoul, 100715, Republic of Korea.
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11
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Increased Potential of Bone Formation with the Intravenous Injection of a Parathyroid Hormone-Related Protein Minicircle DNA Vector. Int J Mol Sci 2021; 22:ijms22169069. [PMID: 34445802 PMCID: PMC8396456 DOI: 10.3390/ijms22169069] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Revised: 08/14/2021] [Accepted: 08/18/2021] [Indexed: 12/14/2022] Open
Abstract
Osteoporosis is commonly treated via the long-term usage of anti-osteoporotic agents; however, poor drug compliance and undesirable side effects limit their treatment efficacy. The parathyroid hormone-related protein (PTHrP) is essential for normal bone formation and remodeling; thus, may be used as an anti-osteoporotic agent. Here, we developed a platform for the delivery of a single peptide composed of two regions of the PTHrP protein (1–34 and 107–139); mcPTHrP 1–34+107–139 using a minicircle vector. We also transfected mcPTHrP 1–34+107–139 into human mesenchymal stem cells (MSCs) and generated Thru 1–34+107–139-producing engineered MSCs (eMSCs) as an alternative delivery system. Osteoporosis was induced in 12-week-old C57BL/6 female mice via ovariectomy. The ovariectomized (OVX) mice were then treated with the two systems; (1) mcPTHrP 1–34+107–139 was intravenously administered three times (once per week); (2) eMSCs were intraperitoneally administered twice (on weeks four and six). Compared with the control OVX mice, the mcPTHrP 1–34+107–139-treated group showed better trabecular bone structure quality, increased bone formation, and decreased bone resorption. Similar results were observed in the eMSCs-treated OVX mice. Altogether, these results provide experimental evidence to support the potential of delivering PTHrP 1–34+107–139 using the minicircle technology for the treatment of osteoporosis.
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12
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Varkouhi AK, Monteiro APT, Tsoporis JN, Mei SHJ, Stewart DJ, Dos Santos CC. Genetically Modified Mesenchymal Stromal/Stem Cells: Application in Critical Illness. Stem Cell Rev Rep 2021; 16:812-827. [PMID: 32671645 PMCID: PMC7363458 DOI: 10.1007/s12015-020-10000-1] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Critical illnesses including sepsis, acute respiratory distress syndromes, ischemic cardiovascular disorders and acute organ injuries are associated with high mortality, morbidity as well as significant health care system expenses. While these diverse conditions require different specific therapeutic approaches, mesenchymal stem/stromal cell (MSCs) are multipotent cells capable of self-renewal, tri-lineage differentiation with a broad range regenerative and immunomodulatory activities, making them attractive for the treatment of critical illness. The therapeutic effects of MSCs have been extensively investigated in several pre-clinical models of critical illness as well as in phase I and II clinical cell therapy trials with mixed results. Whilst these studies have demonstrated the therapeutic potential for MSC therapy in critical illness, optimization for clinical use is an ongoing challenge. MSCs can be readily genetically modified by application of different techniques and tools leading to overexpress or inhibit genes related to their immunomodulatory or regenerative functions. Here we will review recent approaches designed to enhance the therapeutic potential of MSCs with an emphasis on the technology used to generate genetically modified cells, target genes, target diseases and the implication of genetically modified MSCs in cell therapy for critical illness.
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Affiliation(s)
- Amir K Varkouhi
- Department of Chemistry and Environmental Science, New Jersey Institute of Technology (NJIT), Newark, NJ, 07102, USA
| | - Ana Paula Teixeira Monteiro
- Keenan and Li Ka Shing Knowledge Institute, University Health Toronto - St. Michael's Hospital, Toronto, Ontario, Canada.,Institute of Medical Sciences and Department of Medicine, University of Toronto, Toronto, Ontario, Canada
| | - James N Tsoporis
- Keenan and Li Ka Shing Knowledge Institute, University Health Toronto - St. Michael's Hospital, Toronto, Ontario, Canada
| | - Shirley H J Mei
- Ottawa Hospital Research Institute and the University of Ottawa, Ottawa, ON, Canada
| | - Duncan J Stewart
- Ottawa Hospital Research Institute and the University of Ottawa, Ottawa, ON, Canada
| | - Claudia C Dos Santos
- Keenan and Li Ka Shing Knowledge Institute, University Health Toronto - St. Michael's Hospital, Toronto, Ontario, Canada. .,Interdepartmental Division of Critical Care, St. Michael's Hospital/University of Toronto, 30 Bond Street, Room 4-008, Toronto, ON, M5B 1WB, Canada.
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13
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Borrelli MA, Turnquist HR, Little SR. Biologics and their delivery systems: Trends in myocardial infarction. Adv Drug Deliv Rev 2021; 173:181-215. [PMID: 33775706 PMCID: PMC8178247 DOI: 10.1016/j.addr.2021.03.014] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Revised: 03/14/2021] [Accepted: 03/20/2021] [Indexed: 02/07/2023]
Abstract
Cardiovascular disease is the leading cause of death around the world, in which myocardial infarction (MI) is a precipitating event. However, current therapies do not adequately address the multiple dysregulated systems following MI. Consequently, recent studies have developed novel biologic delivery systems to more effectively address these maladies. This review utilizes a scientometric summary of the recent literature to identify trends among biologic delivery systems designed to treat MI. Emphasis is placed on sustained or targeted release of biologics (e.g. growth factors, nucleic acids, stem cells, chemokines) from common delivery systems (e.g. microparticles, nanocarriers, injectable hydrogels, implantable patches). We also evaluate biologic delivery system trends in the entire regenerative medicine field to identify emerging approaches that may translate to the treatment of MI. Future developments include immune system targeting through soluble factor or chemokine delivery, and the development of advanced delivery systems that facilitate the synergistic delivery of biologics.
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Affiliation(s)
- Matthew A Borrelli
- Department of Chemical Engineering, University of Pittsburgh, 940 Benedum Hall, 3700 O'Hara Street, Pittsburgh, PA 15213, USA.
| | - Heth R Turnquist
- Starzl Transplantation Institute, 200 Darragh St, Pittsburgh, PA 15213, USA; Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; Department of Immunology, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15213, USA.
| | - Steven R Little
- Department of Chemical Engineering, University of Pittsburgh, 940 Benedum Hall, 3700 O'Hara Street, Pittsburgh, PA 15213, USA; Department of Bioengineering, University of Pittsburgh, 302 Benedum Hall, 3700 O'Hara Street, Pittsburgh, PA 15213, USA; Department of Clinical and Translational Science, University of Pittsburgh, Forbes Tower, Suite 7057, Pittsburgh, PA 15213, USA; McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219, USA; Department of Immunology, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15213, USA; Department of Pharmaceutical Science, University of Pittsburgh, 3501 Terrace Street, Pittsburgh, PA 15213, USA; Department of Ophthalmology, University of Pittsburgh, 203 Lothrop Street, Pittsburgh, PA 15213, USA.
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14
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Liu L, Gao H, Guo C, Liu T, Li N, Qian Q. Therapeutic Mechanism of Nucleic Acid Drugs. ChemistrySelect 2021. [DOI: 10.1002/slct.202002901] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Affiliation(s)
- Lianxiao Liu
- Nucleic Acid Drug Division Shanghai Cell Therapy Group Co., Ltd. 75 A Qianyang Rd, Jiading District Shanghai 201805 China
| | - Haixia Gao
- Nucleic Acid Drug Division Shanghai Cell Therapy Group Co., Ltd. 75 A Qianyang Rd, Jiading District Shanghai 201805 China
| | - Chuanxin Guo
- Nucleic Acid Drug Division Shanghai Cell Therapy Group Co., Ltd. 75 A Qianyang Rd, Jiading District Shanghai 201805 China
| | - Tao Liu
- Nucleic Acid Drug Division Shanghai Cell Therapy Group Co., Ltd. 75 A Qianyang Rd, Jiading District Shanghai 201805 China
| | - Ning Li
- Nucleic Acid Drug Division Shanghai Cell Therapy Group Co., Ltd. 75 A Qianyang Rd, Jiading District Shanghai 201805 China
| | - Qijun Qian
- Nucleic Acid Drug Division Shanghai Cell Therapy Group Co., Ltd. 75 A Qianyang Rd, Jiading District Shanghai 201805 China
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15
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Peptides as a material platform for gene delivery: Emerging concepts and converging technologies. Acta Biomater 2020; 117:40-59. [PMID: 32966922 DOI: 10.1016/j.actbio.2020.09.027] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2020] [Revised: 08/27/2020] [Accepted: 09/16/2020] [Indexed: 02/06/2023]
Abstract
Successful gene therapies rely on methods that safely introduce DNA into target cells and enable subsequent expression of proteins. To that end, peptides are an attractive materials platform for DNA delivery, facilitating condensation into nanoparticles, delivery into cells, and subcellular release to enable protein expression. Peptides are programmable materials that can be designed to address biocompatibility, stability, and subcellular barriers that limit efficiency of non-viral gene delivery systems. This review focuses on fundamental structure-function relationships regarding peptide design and their impact on nanoparticle physical properties, biologic activity, and biocompatibility. Recent peptide technologies utilize multi-dimensional structures, non-natural chemistries, and combinations of peptides with lipids to achieve desired properties and efficient transfection. Advances in DNA cargo design are also presented to highlight further opportunities for peptide-based gene delivery. Modern DNA designs enable prolonged expression compared to traditional plasmids, providing an additional component that can be synergized with peptide carriers for improved transfection. Peptide transfection systems are poised to become a flexible and efficient platform incorporating new chemistries, functionalities, and improved DNA cargos to usher in a new era of gene therapy.
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Abstract
The current COVID-19 pandemic has substantially accelerated the demands for efficient vaccines. A wide spectrum of approaches includes live attenuated and inactivated viruses, protein subunits and peptides, viral vector-based delivery, DNA plasmids, and synthetic mRNA. Preclinical studies have demonstrated robust immune responses, reduced viral loads and protection against challenges with SARS-CoV-2 in rodents and primates. Vaccine candidates based on all delivery systems mentioned above have been subjected to clinical trials in healthy volunteers. Phase I clinical trials have demonstrated in preliminary findings good safety and tolerability. Evaluation of immune responses in a small number of individuals has demonstrated similar or superior levels of neutralizing antibodies in comparison to immunogenicity detected in COVID-19 patients. Both adenovirus- and mRNA-based vaccines have entered phase II and study protocols for phase III trials with 30,000 participants have been finalized.
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17
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Khoury MK, Stranz AR, Liu B. Pathophysiology of Aortic Aneurysms: Insights from Animal Studies. CARDIOLOGY AND CARDIOVASCULAR MEDICINE 2020; 4:498-514. [PMID: 32968712 PMCID: PMC7508467 DOI: 10.26502/fccm.92920146] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Aortic aneurysms are defined as dilations of the aorta greater than 50 percent. Currently, the only effective treatment for aortic aneurysms is surgical repair, which is recommended only to those that meet criteria. There is no available pharmaceutical therapy to slow aneurysm growth and thus prevent lethal rupture. The development of a number of murine models has allowed in depth studies of various cellular and extracellular components of aneurysm pathophysiology. The identification of key therapeutic targets has resulted in several clinical trials evaluating pharmaceutical candidates to treat aneurysm progression. In this review, we focus on providing recent updates on developments in murine models of aortic aneurysm. In addition, we discuss recent studies of the various cellular and extracellular components of the aorta along with the abutting aortic structures that contribute to aneurysm development and progression.
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Affiliation(s)
- Mitri K Khoury
- Department of Surgery, Division of Vascular and Endovascular Surgery, University of Texas Southwestern Medical Center, Dallas, United States
| | - Amelia R Stranz
- Department of Surgery, Division of Vascular Surgery, University of Wisconsin-Madison, WI, United States
| | - Bo Liu
- Department of Surgery, Division of Vascular Surgery, University of Wisconsin-Madison, WI, United States
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18
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Giménez CS, Castillo MG, Simonin JA, Núñez Pedrozo CN, Pascuali N, Bauzá MDR, Locatelli P, López AE, Belaich MN, Mendiz AO, Crottogini AJ, Cuniberti LA, Olea FD. Effect of intramuscular baculovirus encoding mutant hypoxia-inducible factor 1-alpha on neovasculogenesis and ischemic muscle protection in rabbits with peripheral arterial disease. Cytotherapy 2020; 22:563-572. [PMID: 32723595 DOI: 10.1016/j.jcyt.2020.06.010] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Revised: 06/07/2020] [Accepted: 06/26/2020] [Indexed: 01/09/2023]
Abstract
BACKGROUND AIMS Peripheral arterial disease (PAD) is a progressive, disabling ailment for which no effective treatment exists. Gene therapy-mediated neovascularization has emerged as a potentially useful strategy. We tested the angiogenic and arteriogenic efficacy and safety of a baculovirus (BV) encoding mutant, oxygen-resistant hypoxia-inducible factor 1-alpha (mHIF-1α), in rabbits with PAD. METHODS After assessing the transfection efficiency of the BV.mHIF-1α vector and its tubulogenesis potential in vitro, we randomized rabbits with experimental PAD to receive 1 × 109 copies of BV.mHIF-1α or BV.null (n = 6 per group) 7 days after surgery. Two weeks post-treatment, collateralization (digital angiography) and capillary and arteriolar densities (immunohistochemistry) were measured in the posterior limbs. Ischemic damage was evaluated in adductor and gastrocnemius muscle samples. Tracking of viral DNA in injected zones and remote tissues at different time points was performed in additional rabbits using a BV encoding GFP. RESULTS Angiographically visible collaterals were more numerous in BV.mHIF-1α-treated rabbits (8.12 ± 0.42 vs 6.13 ± 1.15 collaterals/cm2, P < 0.05). The same occurred with arteriolar (27.9 ± 7.0 vs 15.3 ± 4.0 arterioles/mm2) and capillary (341.8 ± 109.9 vs 208.8 ± 87.7 capillaries/mm2, P < 0.05) densities. BV.mHIF-1α-treated rabbits displayed less ischemic muscle damage than BV.null-treated animals. Viral DNA and GFP mRNA were detectable only at 3 and 7 days after injection in hind limbs. Neither the virus nor GFP mRNA was detected in remote tissues. CONCLUSIONS In rabbits with PAD, BV.mHIF-1α induced neovascularization and reduced ischemic damage, exhibiting a good safety profile at 14 days post-treatment. Complementary studies to evaluate its potential usefulness in the clinic are needed.
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Affiliation(s)
- Carlos S Giménez
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina
| | - Martha G Castillo
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina
| | - Jorge A Simonin
- Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Quilmes, Bernal, Argentina
| | - Cristian N Núñez Pedrozo
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina
| | - Natalia Pascuali
- Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - María Del Rosario Bauzá
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina
| | - Paola Locatelli
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina
| | - Ayelén E López
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina
| | - Mariano N Belaich
- Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Quilmes, Bernal, Argentina
| | - Alfredo O Mendiz
- Hospital Universitario de la Fundación Favaloro, Buenos Aires, Argentina
| | - Alberto J Crottogini
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina
| | - Luis A Cuniberti
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina
| | - Fernanda D Olea
- Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina.
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19
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Zimmermann A, Hercher D, Regner B, Frischer A, Sperger S, Redl H, Hacobian A. Evaluation of BMP-2 Minicircle DNA for Enhanced Bone Engineering and Regeneration. Curr Gene Ther 2020; 20:55-63. [PMID: 32338217 DOI: 10.2174/1566523220666200427121350] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Revised: 04/06/2020] [Accepted: 04/08/2020] [Indexed: 12/12/2022]
Abstract
BACKGROUND To date, the significant osteoinductive potential of bone morphogenetic protein 2 (BMP-2) non-viral gene therapy cannot be fully exploited therapeutically. This is mainly due to weak gene delivery and brief expression peaks restricting the therapeutic effect. OBJECTIVE Our objective was to test the application of minicircle DNA, allowing prolonged expression potential. It offers notable advantages over conventional plasmid DNA. The lack of bacterial sequences and the resulting reduction in size, enables safe usage and improved performance for tissue regeneration. METHODS We inserted an optimized BMP-2 gene cassette with minicircle plasmid technology. BMP-2 minicircle plasmids were produced in E. coli yielding plasmids lacking bacterial backbone elements. Comparative studies of these BMP-2 minicircles and conventional BMP-2 plasmids were performed in vitro in cell systems, including bone marrow derived stem cells. Tests performed included gene expression profiles and cell differentiation assays. RESULTS A C2C12 cell line transfected with the BMP-2-Advanced minicircle showed significantly elevated expression of osteocalcin, alkaline phosphatase (ALP) activity, and BMP-2 protein amount when compared to cells transfected with conventional BMP-2-Advanced plasmid. Furthermore, the plasmids show suitability for stem cell approaches by showing significantly higher levels of ALP activity and mineralization when introduced into human bone marrow stem cells (BMSCs). CONCLUSION We have designed a highly bioactive BMP-2 minicircle plasmid with the potential to fulfil clinical requirements for non-viral gene therapy in the field of bone regeneration.
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Affiliation(s)
- Alice Zimmermann
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - David Hercher
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Benedikt Regner
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Amelie Frischer
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Simon Sperger
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Heinz Redl
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
| | - Ara Hacobian
- Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
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20
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Chondrogenic Differentiation from Induced Pluripotent Stem Cells Using Non-Viral Minicircle Vectors. Cells 2020; 9:cells9030582. [PMID: 32121522 PMCID: PMC7140457 DOI: 10.3390/cells9030582] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2019] [Revised: 02/18/2020] [Accepted: 02/27/2020] [Indexed: 12/15/2022] Open
Abstract
Human degenerative cartilage has low regenerative potential. Chondrocyte transplantation offers a promising strategy for cartilage treatment and regeneration. Currently, chondrogenesis using human pluripotent stem cells (hiPSCs) is accomplished using human recombinant growth factors. Here, we differentiate hiPSCs into chondrogenic pellets using minicircle vectors. Minicircles are a non-viral gene delivery system that can produce growth factors without integration into the host genome. We generated minicircle vectors containing bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 3 (TGFβ3) and delivered them to mesenchymal stem cell-like, hiPSC-derived outgrowth (OG) cells. Cell pellets generated using minicircle-transfected OG cells successfully differentiated into the chondrogenic lineage. The implanted minicircle-based chondrogenic pellets recovered the osteochondral defects in rat models. This work is a proof-of-concept study that describes the potential application of minicircle vectors in cartilage regeneration using hiPSCs.
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21
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Barnea-Cramer AO, Singh M, Fischer D, De Silva S, McClements ME, Barnard AR, MacLaren RE. Repair of Retinal Degeneration following Ex Vivo Minicircle DNA Gene Therapy and Transplantation of Corrected Photoreceptor Progenitors. Mol Ther 2020; 28:830-844. [PMID: 32027843 DOI: 10.1016/j.ymthe.2020.01.023] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Revised: 01/07/2020] [Accepted: 01/07/2020] [Indexed: 10/25/2022] Open
Abstract
The authors describe retinal reconstruction and restoration of visual function in heritably blind mice missing the rhodopsin gene using a novel method of ex vivo gene therapy and cell transplantation. Photoreceptor precursors with the same chromosomal genetic mutation were treated ex vivo using minicircle DNA, a non-viral technique that does not present the packaging limitations of adeno-associated virus (AAV) vectors. Following transplantation, genetically modified cells reconstructed a functional retina and supported vision in blind mice harboring the same founder gene mutation. Gene delivery by minicircles showed comparable long-term efficiency to AAV in delivering the missing gene, representing the first non-viral system for robust treatment of photoreceptors. This important proof-of-concept finding provides an innovative convergence of cell and gene therapies for the treatment of hereditary neurodegenerative disease and may be applied in future studies toward ex vivo correction of patient-specific cells to provide an autologous source of tissue to replace lost photoreceptors in inherited retinal blindness. This is the first report using minicircles in photoreceptor progenitors and the first to transplant corrected photoreceptor precursors to restore vision in blind animals.
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Affiliation(s)
| | - Mandeep Singh
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
| | - Dominik Fischer
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; University Eye Hospital and Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany
| | - Samantha De Silva
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; Oxford University Hospitals NHS Foundation Trust, NIHR Oxford Biomedical Research Centre, Oxford, UK
| | | | - Alun R Barnard
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK
| | - Robert E MacLaren
- Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; Oxford University Hospitals NHS Foundation Trust, NIHR Oxford Biomedical Research Centre, Oxford, UK.
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Kanada M, Kim BD, Hardy JW, Ronald JA, Bachmann MH, Bernard MP, Perez GI, Zarea AA, Ge TJ, Withrow A, Ibrahim SA, Toomajian V, Gambhir SS, Paulmurugan R, Contag CH. Microvesicle-Mediated Delivery of Minicircle DNA Results in Effective Gene-Directed Enzyme Prodrug Cancer Therapy. Mol Cancer Ther 2019; 18:2331-2342. [PMID: 31451563 DOI: 10.1158/1535-7163.mct-19-0299] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Revised: 06/13/2019] [Accepted: 08/14/2019] [Indexed: 12/14/2022]
Abstract
An emerging approach for cancer treatment employs the use of extracellular vesicles, specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of codelivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.
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Affiliation(s)
- Masamitsu Kanada
- Department of Pediatrics, Stanford University, Stanford, California. .,Department of Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California.,Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan.,Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan
| | - Bryan D Kim
- Deptartment of Chemistry, University of California, Santa Cruz, California
| | - Jonathan W Hardy
- Department of Pediatrics, Stanford University, Stanford, California.,Department of Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California.,Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan.,Department of Microbiology & Molecular Genetics, Michigan State University, East Lansing, Michigan
| | - John A Ronald
- Department of Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California.,Department of Radiology, Stanford University, Stanford, California.,Robarts Research Institute, Western University, London, Ontario, Canada.,Lawson Health Research Institute, London, Ontario, Canada
| | - Michael H Bachmann
- Department of Pediatrics, Stanford University, Stanford, California.,Department of Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California.,Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan.,Department of Microbiology & Molecular Genetics, Michigan State University, East Lansing, Michigan
| | - Matthew P Bernard
- Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan.,Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan
| | - Gloria I Perez
- Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan
| | - Ahmed A Zarea
- Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan
| | - T Jessie Ge
- Department of Radiology, Stanford University, Stanford, California
| | - Alicia Withrow
- Center for Advanced Microscopy, Michigan State University, East Lansing, Michigan
| | - Sherif A Ibrahim
- Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan.,Deptartment of Histology and Cell Biology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
| | - Victoria Toomajian
- Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan.,Department of Biomedical Engineering, Michigan State University, East Lansing, Michigan
| | - Sanjiv S Gambhir
- Department of Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California.,Department of Radiology, Stanford University, Stanford, California.,Department of Bioengineering, Stanford University, Stanford, California.,Department of Materials Science, Stanford University, Stanford, California
| | - Ramasamy Paulmurugan
- Department of Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California. .,Department of Radiology, Stanford University, Stanford, California
| | - Christopher H Contag
- Department of Pediatrics, Stanford University, Stanford, California. .,Department of Molecular Imaging Program at Stanford (MIPS), Stanford University, Stanford, California.,Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, Michigan.,Department of Microbiology & Molecular Genetics, Michigan State University, East Lansing, Michigan.,Department of Biomedical Engineering, Michigan State University, East Lansing, Michigan
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23
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Han D, Wang Y, Chen J, Zhang J, Yu P, Zhang R, Li S, Tao B, Wang Y, Qiu Y, Xu M, Gao E, Cao F. Activation of melatonin receptor 2 but not melatonin receptor 1 mediates melatonin-conferred cardioprotection against myocardial ischemia/reperfusion injury. J Pineal Res 2019; 67:e12571. [PMID: 30903623 DOI: 10.1111/jpi.12571] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Revised: 03/01/2019] [Accepted: 03/17/2019] [Indexed: 12/14/2022]
Abstract
Accumulated pieces of evidence have proved the beneficial effects of melatonin on myocardial ischemia/reperfusion (MI/R) injury, and these effects were largely dependent on melatonin membrane receptor activation. In humans and other mammals, there are two types of melatonin receptors, including the melatonin receptor 1 (MT1, melatonin receptor 1a or MTNR1A) and melatonin receptor 1 (MT2, melatonin receptor 1b or MTNR1B) receptor subtypes. However, which receptor mediates melatonin-conferred cardioprotection remains unclear. In this study, we employed both loss-of-function and gain-of-function approaches to reveal the answer. Mice (wild-type; MT1 or MT2 silencing by in vivo minicircle vector; and those overexpressing MT1 or MT2 by in vivo AAV9 vector) were exposed to MI/R injury. Both MT1 and MT2 were present in wild-type myocardium. MT2, but not MT1, was essentially upregulated after MI/R Melatonin administration significantly reduced myocardial injury and improved cardiac function after MI/R Mechanistically, melatonin treatment suppressed MI/R-initiated myocardial oxidative stress and nitrative stress, alleviated endoplasmic reticulum stress and mitochondrial injury, and inhibited myocardial apoptosis. These beneficial actions of melatonin were absent in MT2-silenced heart, but not the MT1 subtype. Furthermore, AAV9-mediated cardiomyocyte-specific overexpression of MT2, but not MT1, mitigated MI/R injury and improved cardiac dysfunction, which was accompanied by significant amelioration of oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction. Mechanistically, MT2 protected primary cardiomyocytes against hypoxia/reoxygenation injury via MT2/Notch1/Hes1/RORα signaling. Our study presents the first direct evidence that the MT2 subtype, but not MT1, is a novel endogenous cardiac protective receptor against MI/R injury. Medications specifically targeting MT2 may hold promise in fighting ischemic heart disease.
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MESH Headings
- Animals
- Apoptosis
- Disease Models, Animal
- Endoplasmic Reticulum Stress/genetics
- Humans
- Male
- Mice
- Myocardial Reperfusion Injury/genetics
- Myocardial Reperfusion Injury/metabolism
- Myocardial Reperfusion Injury/pathology
- Myocardial Reperfusion Injury/prevention & control
- Myocardium/metabolism
- Myocardium/pathology
- Myocytes, Cardiac/metabolism
- Myocytes, Cardiac/pathology
- Oxidative Stress/genetics
- Receptor, Melatonin, MT1/genetics
- Receptor, Melatonin, MT1/metabolism
- Receptor, Melatonin, MT2/genetics
- Receptor, Melatonin, MT2/metabolism
- Signal Transduction
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Affiliation(s)
- Dong Han
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
| | - Yongjun Wang
- Department of Cardiovascular Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jiangwei Chen
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
| | - Jibin Zhang
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
| | - Peng Yu
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
| | - Ran Zhang
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Shuang Li
- Department of Cardiology, Chengdu Military General Hospital, Chengdu, China
| | - Bo Tao
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Yabin Wang
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Ya Qiu
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Mengqi Xu
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
| | - Erhe Gao
- Center for Translational Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania
| | - Feng Cao
- Department of Cardiology, National Clinical Research Center for Geriatric Diseases, 2nd Medical Center, Chinese PLA General Hospital, Beijing, China
- Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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24
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Adam M, Kooreman NG, Jagger A, Wagenhäuser MU, Mehrkens D, Wang Y, Kayama Y, Toyama K, Raaz U, Schellinger IN, Maegdefessel L, Spin JM, Hamming JF, Quax PHA, Baldus S, Wu JC, Tsao PS. Systemic Upregulation of IL-10 (Interleukin-10) Using a Nonimmunogenic Vector Reduces Growth and Rate of Dissecting Abdominal Aortic Aneurysm. Arterioscler Thromb Vasc Biol 2019; 38:1796-1805. [PMID: 29880489 DOI: 10.1161/atvbaha.117.310672] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Objective- Recruitment of immunologic competent cells to the vessel wall is a crucial step in formation of abdominal aortic aneurysms (AAA). Innate immunity effectors (eg, macrophages), as well as mediators of adaptive immunity (eg, T cells), orchestrate a local vascular inflammatory response. IL-10 (interleukin-10) is an immune-regulatory cytokine with a crucial role in suppression of inflammatory processes. We hypothesized that an increase in systemic IL-10-levels would mitigate AAA progression. Approach and Results- Using a single intravenous injection protocol, we transfected an IL-10 transcribing nonimmunogenic minicircle vector into the Ang II (angiotensin II)-ApoE-/- infusion mouse model of AAA. IL-10 minicircle transfection significantly reduced average aortic diameter measured via ultrasound at day 28 from 166.1±10.8% (control) to 131.0±5.8% (IL-10 transfected). Rates of dissecting AAA were reduced by IL-10 treatment, with an increase in freedom from dissecting AAA from 21.5% to 62.3%. Using flow cytometry of aortic tissue from minicircle IL-10-treated animals, we found a significantly higher percentage of CD4+/CD25+/Foxp3 (forkhead box P3)+ regulatory T cells, with fewer CD8+/GZMB+ (granzyme B) cytotoxic T cells. Furthermore, isolated aortic macrophages produced less TNF-α (tumor necrosis factor-α), more IL-10, and were more likely to be MRC1 (mannose receptor, C type 1)-positive alternatively activated macrophages. These results concurred with gene expression analysis of lipopolysaccharide-stimulated and Ang II-primed human peripheral blood mononuclear cells. Conclusions- Taken together, we provide an effective gene therapy approach to AAA in mice by enhancing antiinflammatory and dampening proinflammatory pathways through minicircle-induced augmentation of systemic IL-10 expression.
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Affiliation(s)
- Matti Adam
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,Department of Cardiovascular Medicine, Cologne Cardiovascular Research Center, University of Cologne, University Heart Center, Germany (M.A., D.M., S.B.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.)
| | - Nigel Geoffrey Kooreman
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,Department of Vascular Surgery, Leiden University Medical Center, The Netherlands (N.G.K., J.F.H., P.H.A.Q.)
| | - Ann Jagger
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.)
| | - Markus U Wagenhäuser
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.)
| | - Dennis Mehrkens
- Department of Cardiovascular Medicine, Cologne Cardiovascular Research Center, University of Cologne, University Heart Center, Germany (M.A., D.M., S.B.)
| | - Yongming Wang
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.)
| | - Yosuke Kayama
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.)
| | - Kensuke Toyama
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.)
| | - Uwe Raaz
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.).,Heart Center, Georg-August-University Göttingen, Germany (U.R., I.N.S.)
| | - Isabel N Schellinger
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.).,Heart Center, Georg-August-University Göttingen, Germany (U.R., I.N.S.)
| | - Lars Maegdefessel
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,Department of Medicine, Karolinska Institutet, Stockholm, Sweden (L.M.)
| | - Joshua M Spin
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.)
| | - Jaap F Hamming
- Department of Vascular Surgery, Leiden University Medical Center, The Netherlands (N.G.K., J.F.H., P.H.A.Q.)
| | - Paul H A Quax
- Department of Vascular Surgery, Leiden University Medical Center, The Netherlands (N.G.K., J.F.H., P.H.A.Q.)
| | - Stephan Baldus
- Department of Cardiovascular Medicine, Cologne Cardiovascular Research Center, University of Cologne, University Heart Center, Germany (M.A., D.M., S.B.)
| | - Joseph C Wu
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.)
| | - Philip S Tsao
- From the Division of Cardiovascular Medicine, Cardiovascular Institute, Stanford University School of Medicine, CA (M.A., N.G.K., A.J., M.U.W., Y.W., YK., K.T., U.R., I.N.S., L.M., J.M.S., J.C.W., P.S.T.).,VA Palo Alto Health Care System, CA (M.A., A.J., M.U.W., Y.K., K.T., U.R., I.N.S., J.M.S., P.S.T.)
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25
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Lim SW, Shin YJ, Luo K, Quan Y, Ko EJ, Chung BH, Yang CW. Host cell in vivo production of the synthetic drug anti-CD25/IL-10 using minicircle vector. FASEB J 2019; 33:10889-10901. [PMID: 31266358 DOI: 10.1096/fj.201900833r] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Synthetic biologic drugs are highly successful for induction therapy in transplantation, but the development of novel biologics is limited because of the high cost of synthesis and purification. In this study, we developed a novel strategy for the production of synthetic protein drugs in vivo by the host itself. We utilized minicircle (MC) technology, which can robustly express a target molecule and secrete it from cells, as an indirect method to produce a protein of interest in vivo. We designed an MC vector containing the sequences of basiliximab (anti-CD25 mAb) and IL-10. We verified the substantial production of the anti-CD25/IL-10 protein from the MC in vitro and in vivo. The therapeutic effect of MC-derived anti-CD25/IL-10 was evaluated in a skin allograft mouse model by single intravenous infusion. Mice treated with the MC encoding anti-CD25/IL-10 exhibited prolonged skin allograft survival times accompanied by improved histologic changes and immunologic regulation. These findings indicate that the anti-CD25/IL-10 protein drug obtained by MC technology is functionally active and relevant for reducing allograft rejection. This self-reproducible strategy for synthetic protein drugs using MCs is a promising tool for transplantation.-Lim, S. W., Shin, Y. J., Luo, K., Quan, Y., Ko, E. J., Chung, B. H., Yang, C. W. Host cell in vivo production of the synthetic drug anti-CD25/IL-10 using minicircle vector.
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Affiliation(s)
- Sun Woo Lim
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Yoo Jin Shin
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Kang Luo
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Yi Quan
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Eun Jeong Ko
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Division of Nephrology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Byung Ha Chung
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Division of Nephrology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Chul Woo Yang
- Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Transplant Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea.,Division of Nephrology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea
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26
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Lee J, Arun Kumar S, Jhan YY, Bishop CJ. Engineering DNA vaccines against infectious diseases. Acta Biomater 2018; 80:31-47. [PMID: 30172933 PMCID: PMC7105045 DOI: 10.1016/j.actbio.2018.08.033] [Citation(s) in RCA: 129] [Impact Index Per Article: 18.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2018] [Revised: 08/14/2018] [Accepted: 08/23/2018] [Indexed: 12/30/2022]
Abstract
Engineering vaccine-based therapeutics for infectious diseases is highly challenging, as trial formulations are often found to be nonspecific, ineffective, thermally or hydrolytically unstable, and/or toxic. Vaccines have greatly improved the therapeutic landscape for treating infectious diseases and have significantly reduced the threat by therapeutic and preventative approaches. Furthermore, the advent of recombinant technologies has greatly facilitated growth within the vaccine realm by mitigating risks such as virulence reversion despite making the production processes more cumbersome. In addition, seroconversion can also be enhanced by recombinant technology through kinetic and nonkinetic approaches, which are discussed herein. Recombinant technologies have greatly improved both amino acid-based vaccines and DNA-based vaccines. A plateau of interest has been reached between 2001 and 2010 for the scientific community with regard to DNA vaccine endeavors. The decrease in interest may likely be attributed to difficulties in improving immunogenic properties associated with DNA vaccines, although there has been research demonstrating improvement and optimization to this end. Despite improvement, to the extent of our knowledge, there are currently no regulatory body-approved DNA vaccines for human use (four vaccines approved for animal use). This article discusses engineering DNA vaccines against infectious diseases while discussing advantages and disadvantages of each, with an emphasis on applications of these DNA vaccines. Statement of Significance This review paper summarizes the state of the engineered/recombinant DNA vaccine field, with a scope entailing “Engineering DNA vaccines against infectious diseases”. We endeavor to emphasize recent advances, recapitulating the current state of the field. In addition to discussing DNA therapeutics that have already been clinically translated, this review also examines current research developments, and the challenges thwarting further progression. Our review covers: recombinant DNA-based subunit vaccines; internalization and processing; enhancing immune protection via adjuvants; manufacturing and engineering DNA; the safety, stability and delivery of DNA vaccines or plasmids; controlling gene expression using plasmid engineering and gene circuits; overcoming immunogenic issues; and commercial successes. We hope that this review will inspire further research in DNA vaccine development.
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27
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Makhija H, Roy S, Hoon S, Ghadessy FJ, Wong D, Jaiswal R, Campana D, Dröge P. A novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expression. Nucleic Acids Res 2018; 46:e99. [PMID: 29893931 PMCID: PMC6144826 DOI: 10.1093/nar/gky500] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2017] [Accepted: 05/22/2018] [Indexed: 02/06/2023] Open
Abstract
Advances in stem cell engineering, gene therapy and molecular medicine often involve genome engineering at a cellular level. However, functionally large or multi transgene cassette insertion into the human genome still remains a challenge. Current practices such as random transgene integration or targeted endonuclease-based genome editing are suboptimal and might pose safety concerns. Taking this into consideration, we previously developed a transgenesis tool derived from phage λ integrase (Int) that precisely recombines large plasmid DNA into an endogenous sequence found in human Long INterspersed Elements-1 (LINE-1). Despite this advancement, biosafety concerns associated with bacterial components of plasmids, enhanced uptake and efficient transgene expression remained problematic. We therefore further improved and herein report a more superior Int-based transgenesis tool. This novel Int platform allows efficient and easy derivation of sufficient amounts of seamless supercoiled transgene vectors from conventional plasmids via intramolecular recombination as well as subsequent intermolecular site-specific genome integration into LINE-1. Furthermore, we identified certain LINE-1 as preferred insertion sites for Int-mediated seamless vector transgenesis, and showed that targeted anti-CD19 chimeric antigen receptor gene integration achieves high-level sustained transgene expression in human embryonic stem cell clones for potential downstream therapeutic applications.
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Affiliation(s)
- Harshyaa Makhija
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
| | - Suki Roy
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
| | - Shawn Hoon
- Molecular Engineering Lab, Biomedical Sciences Institute, Agency for Science Technology and Research, 61 Biopolis Drive, Singapore 138673
| | | | - Desmond Wong
- Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
| | - Rahul Jaiswal
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
| | - Dario Campana
- Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore 117599
| | - Peter Dröge
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.,Nanyang Institute of Structural Biology, Nanyang Technological University, Experimental Medicine Building (EMB), 59 Nanyang Drive, Singapore 636921
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28
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Youngblood RL, Truong NF, Segura T, Shea LD. It's All in the Delivery: Designing Hydrogels for Cell and Non-viral Gene Therapies. Mol Ther 2018; 26:2087-2106. [PMID: 30107997 PMCID: PMC6127639 DOI: 10.1016/j.ymthe.2018.07.022] [Citation(s) in RCA: 52] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2018] [Revised: 07/24/2018] [Accepted: 07/24/2018] [Indexed: 01/08/2023] Open
Abstract
Hydrogels provide a regenerative medicine platform with their ability to create an environment that supports transplanted or endogenous infiltrating cells and enables these cells to restore or replace the function of tissues lost to disease or trauma. Furthermore, these systems have been employed as delivery vehicles for therapeutic genes, which can direct and/or enhance the function of the transplanted or endogenous cells. Herein, we review recent advances in the development of hydrogels for cell and non-viral gene delivery through understanding the design parameters, including both physical and biological components, on promoting transgene expression, cell engraftment, and ultimately cell function. Furthermore, this review identifies emerging opportunities for combining cell and gene delivery approaches to overcome challenges to the field.
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Affiliation(s)
- Richard L Youngblood
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Norman F Truong
- Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Tatiana Segura
- Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.
| | - Lonnie D Shea
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
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Abou-Saleh H, Zouein FA, El-Yazbi A, Sanoudou D, Raynaud C, Rao C, Pintus G, Dehaini H, Eid AH. The march of pluripotent stem cells in cardiovascular regenerative medicine. Stem Cell Res Ther 2018; 9:201. [PMID: 30053890 PMCID: PMC6062943 DOI: 10.1186/s13287-018-0947-5] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Cardiovascular disease (CVD) continues to be the leading cause of global morbidity and mortality. Heart failure remains a major contributor to this mortality. Despite major therapeutic advances over the past decades, a better understanding of molecular and cellular mechanisms of CVD as well as improved therapeutic strategies for the management or treatment of heart failure are increasingly needed. Loss of myocardium is a major driver of heart failure. An attractive approach that appears to provide promising results in reducing cardiac degeneration is stem cell therapy (SCT). In this review, we describe different types of stem cells, including embryonic and adult stem cells, and we provide a detailed discussion of the properties of induced pluripotent stem cells (iPSCs). We also present and critically discuss the key methods used for converting somatic cells to pluripotent cells and iPSCs to cardiomyocytes (CMs), along with their advantages and limitations. Integrating and non-integrating reprogramming methods as well as characterization of iPSCs and iPSC-derived CMs are discussed. Furthermore, we critically present various methods of differentiating iPSCs to CMs. The value of iPSC-CMs in regenerative medicine as well as myocardial disease modeling and cardiac regeneration are emphasized.
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Affiliation(s)
- Haissam Abou-Saleh
- Department of Biological and Environmental Sciences, Qatar University, Doha, Qatar
| | - Fouad A. Zouein
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Ahmed El-Yazbi
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
- Department of Pharmacology and Toxicology, Alexandria University, Alexandria, Egypt
| | - Despina Sanoudou
- Clinical Genomics and Pharmacogenomics Unit, 4th Department of Internal Medicine, “Attikon” Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | | | - Christopher Rao
- Department of Surgery, Queen Elizabeth Hospital, Woolwich, London, UK
| | - Gianfranco Pintus
- Department of Biomedical Sciences, College of Health Sciences, Qatar University, Doha, Qatar
| | - Hassan Dehaini
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Ali H. Eid
- Department of Biological and Environmental Sciences, Qatar University, Doha, Qatar
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
- Department of Biomedical Sciences, College of Health Sciences, Qatar University, Doha, Qatar
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30
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Cheng C, Tang N, Li J, Cao S, Zhang T, Wei X, Wang H. Bacteria-free minicircle DNA system to generate integration-free CAR-T cells. J Med Genet 2018; 56:10-17. [PMID: 30030293 PMCID: PMC6327863 DOI: 10.1136/jmedgenet-2018-105405] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2018] [Revised: 06/05/2018] [Accepted: 06/18/2018] [Indexed: 12/30/2022]
Abstract
BACKGROUND Chimeric antigen receptor T (CAR-T) cells engineered with lentiviral and retroviral vectors have been successfully applied to treat patients with B cell malignancy. However, viral integration in T cells has the potential risk of mutagenesis, and viral vector production demands effort and is costly. Using non-integrative episomal vector such as minicircle vector to generate integration-free CAR-T cells is an attractive option. METHODS AND RESULTS We established a novel method to generate minicircle vector within a few hours using simple molecular biology techniques. Since no bacteria is involved, we named these vectors bacteria-free (BF) minicircle. In comparison with plasmids, BF minicircle vector enabled higher transgene expression and improved cell viability in human cell line, stem cells and primary T cells. Using BF minicircle vector, we generated integration-free CAR-T cells, which eliminated cancer cells efficiently both in vitro and in vivo. CONCLUSION BF minicircle vector will be useful in basic research as well as in clinical applications such as CAR-T and gene therapy. Although the transgene expression of minicircle vector lasts apparently shorter than that of insertional lentivirus, multiple rounds of BF minicircle CAR-T cell infusion could eliminate cancer cells efficiently. On the other hand, a relatively shorter CAR-T cell persistence provides an opportunity to avoid serious side effects such as cytokine storm or on-target off-tumour toxicity.
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Affiliation(s)
- Chen Cheng
- School of Life Sciences, University of Science and Technology of China, Hefei, China.,State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China
| | - Na Tang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China
| | - Jiaxin Li
- Northwest Agriculture and Forestry University, Yangling, China
| | - Shiwei Cao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China.,University of the Chinese Academy of Sciences, Beijing, China
| | - Tongtong Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China.,University of the Chinese Academy of Sciences, Beijing, China
| | | | - Haoyi Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, China.,University of the Chinese Academy of Sciences, Beijing, China
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31
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Abstract
Stem cell therapy is a promising approach to the treatment of ischemic heart disease via replenishing cell loss after myocardial infarction. Both preclinical studies and clinical trials have indicated that cardiac function improved consistently, but very modestly after cell-based therapy. This mainly attributed to low cell survival rate, engraftment and functional integration, which became the major challenges to regenerative medicine. In recent years, several new cell types have been developed to regenerate cardiomyocytes and novel delivery approaches helped to increase local cell retention. New strategies, such as cell pretreatment, gene-based therapy, tissue engineering, extracellular vesicles application and immunologic regulation, have surged and brought about improved cell survival and functional integration leading to better therapeutic effects after cell transplantation. In this review, we summarize these new strategies targeting at challenges of cardiac regenerative medicine and discuss recent evidences that may hint their effectiveness in the future clinical settings.
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32
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Minicircle Mediated Gene Delivery to Canine and Equine Mesenchymal Stem Cells. Int J Mol Sci 2017; 18:ijms18040819. [PMID: 28417917 PMCID: PMC5412403 DOI: 10.3390/ijms18040819] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2017] [Revised: 04/03/2017] [Accepted: 04/10/2017] [Indexed: 12/31/2022] Open
Abstract
Gene-directed tissue repair offers the clinician, human or veterinary, the chance to enhance cartilage regeneration and repair at a molecular level. Non-viral plasmid vectors have key biosafety advantages over viral vector systems for regenerative therapies due to their episomal integration however, conventional non-viral vectors can suffer from low transfection efficiency. Our objective was to identify and validate in vitro a novel non-viral gene expression vector that could be utilized for ex vivo and in vivo delivery to stromal-derived mesenchymal stem cells (MSCs). Minicircle plasmid DNA vector containing green fluorescent protein (GFP) was generated and transfected into adipose-derived MSCs from three species: canine, equine and rodent and transfection efficiency was determined. Both canine and rat cells showed transfection efficiencies of approximately 40% using minicircle vectors with equine cells exhibiting lower transfection efficiency. A Sox9-expressing minicircle vector was generated and transfected into canine MSCs. Successful transfection of the minicircle-Sox9 vector was confirmed in canine cells by Sox9 immunostaining. This study demonstrate the application and efficacy of a novel non-viral expression vector in canine and equine MSCs. Minicircle vectors have potential use in gene-directed regenerative therapies in non-rodent animal models for treatment of cartilage injury and repair.
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33
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Sequence-Modified Antibiotic Resistance Genes Provide Sustained Plasmid-Mediated Transgene Expression in Mammals. Mol Ther 2017; 25:1187-1198. [PMID: 28365028 DOI: 10.1016/j.ymthe.2017.03.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2016] [Revised: 02/26/2017] [Accepted: 03/02/2017] [Indexed: 02/05/2023] Open
Abstract
Conventional plasmid vectors are incapable of achieving sustained levels of transgene expression in vivo even in quiescent mammalian tissues because the transgene expression cassette is silenced. Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. Here, we show that transcriptional silencing can be substantially forestalled by increasing the An/Tn sequence composition in the plasmid bacterial backbone. Increasing numbers of An/Tn sequences increased sustained transcription of both backbone sequences and adjacent expression cassettes. In order to recapitulate these expression profiles in compact and portable plasmid DNA backbones, we engineered the standard kanamycin or ampicillin antibiotic resistance genes, optimizing the number of An/Tn sequence without altering the encoded amino acids. The resulting vector backbones yield sustained transgene expression from mouse liver, providing generic DNA vectors capable of sustained transgene expression without additional genes or mammalian regulatory elements.
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Liu N, Wang BJ, Broughton KM, Alvarez R, Siddiqi S, Loaiza R, Nguyen N, Quijada P, Gude N, Sussman MA. PIM1-minicircle as a therapeutic treatment for myocardial infarction. PLoS One 2017; 12:e0173963. [PMID: 28323876 PMCID: PMC5360264 DOI: 10.1371/journal.pone.0173963] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2016] [Accepted: 03/01/2017] [Indexed: 01/13/2023] Open
Abstract
PIM1, a pro-survival gene encoding a serine/ threonine kinase, influences cell proliferation and survival. Modification of cardiac progenitor cells (CPCs) or cardiomyocytes with PIM1 using a lentivirus-based delivery method showed long-term improved cardiac function after myocardial infarction (MI). However, lentivirus based delivery methods have stringent FDA regulation with respect to clinical trials. To provide an alternative and low risk PIM1 delivery method, this study examined the use of a non-viral modified plasmid-minicircle (MC) as a vehicle to deliver PIM1 into mouse CPCs (mCPCs) in vitro and the myocardium in vivo. MC containing a turbo gfp reporter gene (gfp-MC) was used as a transfection and injection control. PIM1 was subcloned into gfp-MC (PIM1-MC) and then transfected into mCPCs at an efficiency of 29.4±3.7%. PIM1-MC engineered mCPCs (PIM1-mCPCs) exhibit significantly (P<0.05) better survival rate under oxidative treatment. PIM1-mCPCs also exhibit 1.9±0.1 and 2.2±0.2 fold higher cell proliferation at 3 and 5 days post plating, respectively, as compared to gfp-MC transfected mCPCs control. PIM1-MC was injected directly into ten-week old adult FVB female mice hearts in the border zone immediately after MI. Delivery of PIM1 into myocardium was confirmed by GFP+ cardiomyocytes. Mice with PIM1-MC injection showed increased protection compared to gfp-MC injection groups measured by ejection fraction at 3 and 7 days post injury (P = 0.0379 and P = 0.0262 by t-test, respectively). Success of PIM1 delivery and integration into mCPCs in vitro and cardiomyocytes in vivo by MC highlights the possibility of a non-cell based therapeutic approach for treatment of ischemic heart disease and MI.
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Affiliation(s)
- Nan Liu
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Bingyan J. Wang
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Kathleen M. Broughton
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Roberto Alvarez
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Sailay Siddiqi
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Rebeca Loaiza
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Nicky Nguyen
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Pearl Quijada
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Natalie Gude
- Biology Department, San Diego State University, San Diego, California, United States of America
| | - Mark A. Sussman
- Biology Department, San Diego State University, San Diego, California, United States of America
- * E-mail:
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Šimčíková M, Prather KLJ, Prazeres DMF, Monteiro GA. Towards effective non-viral gene delivery vector. Biotechnol Genet Eng Rev 2017; 31:82-107. [PMID: 27160661 DOI: 10.1080/02648725.2016.1178011] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Despite very good safety records, clinical trials using plasmid DNA failed due to low transfection efficiency and brief transgene expression. Although this failure is both due to poor plasmid design and to inefficient delivery methods, here we will focus on the former. The DNA elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats showed detrimental effects on plasmids' performance as biopharmaceuticals. On the other hand, careful selection of promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences for therapeutic protein expression can enhance the clinical efficacy. Minimal vectors, which are devoid of the bacterial backbone and consist exclusively of the eukaryotic expression cassette, demonstrate better performance in terms of expression levels, bioavailability, transfection rates and increased therapeutic effects. Although the results are promising, minimal vectors have not taken over the conventional plasmids in clinical trials due to challenging manufacturing issues.
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Affiliation(s)
- Michaela Šimčíková
- a MIT-Portugal Program.,b iBB-Institute for Bioengineering and Biosciences , Lisbon , Portugal
| | - Kristala L J Prather
- a MIT-Portugal Program.,c Department of Chemical Engineering , Massachusetts Institute of Technology , Cambridge , MA , USA
| | - Duarte M F Prazeres
- a MIT-Portugal Program.,c Department of Chemical Engineering , Massachusetts Institute of Technology , Cambridge , MA , USA.,d Department of Bioengineering , Instituto Superior Técnico , Lisbon , Portugal
| | - Gabriel A Monteiro
- a MIT-Portugal Program.,c Department of Chemical Engineering , Massachusetts Institute of Technology , Cambridge , MA , USA.,d Department of Bioengineering , Instituto Superior Técnico , Lisbon , Portugal
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36
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Park N, Rim YA, Jung H, Kim J, Yi H, Kim Y, Jang Y, Jung SM, Lee J, Kwok SK, Park SH, Ju JH. Etanercept-Synthesising Mesenchymal Stem Cells Efficiently Ameliorate Collagen-Induced Arthritis. Sci Rep 2017; 7:39593. [PMID: 28084468 PMCID: PMC5234034 DOI: 10.1038/srep39593] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2016] [Accepted: 11/25/2016] [Indexed: 12/18/2022] Open
Abstract
Mesenchymal stem cells (MSCs) have multiple properties including anti-inflammatory and immunomodulatory effects in various disease models and clinical treatments. These beneficial effects, however, are sometimes inconsistent and unpredictable. For wider and proper application, scientists sought to improve MSC functions by engineering. We aimed to invent a novel method to produce synthetic biological drugs from engineered MSCs. We investigated the anti-arthritic effect of engineered MSCs in a collagen-induced arthritis (CIA) model. Biologics such as etanercept are the most successful drugs used in anti-cytokine therapy. Biologics are made of protein components, and thus can be theoretically produced from cells including MSCs. MSCs were transfected with recombinant minicircles encoding etanercept (trade name, Enbrel), which is a tumour necrosis factor α blocker currently used to treat rheumatoid arthritis. We confirmed minicircle expression in MSCs in vitro based on GFP. Etanercept production was verified from the conditioned media. We confirmed that self-reproduced etanercept was biologically active in vitro. Arthritis subsided more efficiently in CIA mice injected with mcTNFR2MSCs than in those injected with conventional MSCs or etanercept only. Although this novel strategy is in a very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics and engineering MSCs.
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Affiliation(s)
- Narae Park
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Yeri Alice Rim
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Hyerin Jung
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Juryun Kim
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Hyoju Yi
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea.,Department of Medicine, Institute for Stem Cell Biology and Regenerative Medicine, and Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA
| | - Youngkyun Kim
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea.,Department of Medicine, Institute for Stem Cell Biology and Regenerative Medicine, and Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA
| | - Yeonsue Jang
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Seung Min Jung
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Jennifer Lee
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Seung-Ki Kwok
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Sung-Hwan Park
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
| | - Ji Hyeon Ju
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Republic of Korea
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Brett E, Zielins ER, Luan A, Ooi CC, Shailendra S, Atashroo D, Menon S, Blackshear C, Flacco J, Quarto N, Wang SX, Longaker MT, Wan DC. Magnetic Nanoparticle-Based Upregulation of B-Cell Lymphoma 2 Enhances Bone Regeneration. Stem Cells Transl Med 2017; 6:151-160. [PMID: 28170185 PMCID: PMC5442739 DOI: 10.5966/sctm.2016-0051] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2016] [Accepted: 06/16/2016] [Indexed: 01/08/2023] Open
Abstract
Clinical translation of cell-based strategies for tissue regeneration remains challenging because survival of implanted cells within hostile, hypoxic wound environments is uncertain. Overexpression of B-cell lymphoma 2 (Bcl-2) has been shown to inhibit apoptosis in implanted cells. The present study describes an "off the shelf" prefabricated scaffold integrated with magnetic nanoparticles (MNPs) used to upregulate Bcl-2 expression in implanted adipose-derived stromal cells for bone regeneration. Iron oxide cores were sequentially coated with branched polyethyleneimine, minicircle plasmid encoding green fluorescent protein and Bcl-2, and poly-β-amino ester. Through in vitro assays, increased osteogenic potential and biological resilience were demonstrated in the magnetofected group over control and nucleofected groups. Similarly, our in vivo calvarial defect study showed that magnetofection had an efficiency rate of 30%, which in turn resulted in significantly more healing compared with control group and nucleofected group. Our novel, prefabricated MNP-integrated scaffold allows for in situ postimplant temporospatial control of cell transfection to augment bone regeneration. Stem Cells Translational Medicine 2017;6:151-160.
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Affiliation(s)
- Elizabeth Brett
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Elizabeth R. Zielins
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Anna Luan
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Chin Chun Ooi
- Department of Material Science Engineering, Stanford University, Stanford, California, USA
| | - Siny Shailendra
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - David Atashroo
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Siddarth Menon
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Charles Blackshear
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - John Flacco
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Natalina Quarto
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Shan X. Wang
- Department of Material Science Engineering, Stanford University, Stanford, California, USA
- Electrical Engineering, Stanford University, Stanford, California, USA
| | - Michael T. Longaker
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA
| | - Derrick C. Wan
- Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California, USA
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Exosomes Derived from Embryonic Stem Cells as Potential Treatment for Cardiovascular Diseases. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 998:187-206. [DOI: 10.1007/978-981-10-4397-0_13] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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39
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Hnatiuk AP, Ong SG, Olea FD, Locatelli P, Riegler J, Lee WH, Jen CH, De Lorenzi A, Giménez CS, Laguens R, Wu JC, Crottogini A. Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant Human Hypoxia-Inducible Factor 1-α (HIF1-α) in an Ovine Model of Acute Myocardial Infarction. J Am Heart Assoc 2016; 5:JAHA.116.003714. [PMID: 27385426 PMCID: PMC5015403 DOI: 10.1161/jaha.116.003714] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Background Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia‐inducible factor 1‐α (HIF1‐α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen‐resistant HIF1‐α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI. Methods and Results Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1‐α (BMMSC‐HIF) were injected in the peri‐infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P<0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2‐fold (P<0.001) in the presence of markedly decreased end‐systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo‐treated animals (n=6), neither parameters changed over time. HIF1‐α‐transfected BMMSCs (BMMSC‐HIF) induced angio‐/arteriogenesis and decreased apoptosis by HIF1‐mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin‐1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long‐term retention of BMMSC‐HIF. Conclusions Intramyocardial delivery of BMMSC‐HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1‐mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.
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Affiliation(s)
- Anna P Hnatiuk
- Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Universidad Favaloro-CONICET, Buenos Aires, Argentina Departamento de Fisiología, Facultad de Ciencias Médicas, Universidad Favaloro, Buenos Aires, Argentina
| | - Sang-Ging Ong
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA
| | - Fernanda D Olea
- Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Universidad Favaloro-CONICET, Buenos Aires, Argentina Departamento de Fisiología, Facultad de Ciencias Médicas, Universidad Favaloro, Buenos Aires, Argentina
| | - Paola Locatelli
- Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Universidad Favaloro-CONICET, Buenos Aires, Argentina Departamento de Fisiología, Facultad de Ciencias Médicas, Universidad Favaloro, Buenos Aires, Argentina
| | - Johannes Riegler
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA
| | - Won Hee Lee
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA
| | | | - Andrea De Lorenzi
- Departmento de Cardiología, Hospital Universitario de la Foundación Favaloro, Buenos Aires, Argentina
| | - Carlos S Giménez
- Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Universidad Favaloro-CONICET, Buenos Aires, Argentina Departamento de Fisiología, Facultad de Ciencias Médicas, Universidad Favaloro, Buenos Aires, Argentina
| | - Rubén Laguens
- Departmento de Patología, Facultad de Ciencias Médicas, Universidad Favaloro, Buenos Aires, Argentina
| | - Joseph C Wu
- Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA
| | - Alberto Crottogini
- Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Universidad Favaloro-CONICET, Buenos Aires, Argentina Departamento de Fisiología, Facultad de Ciencias Médicas, Universidad Favaloro, Buenos Aires, Argentina
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Van Overstraeten-Schlögel N, Ho-Shim Y, Tevel V, Bontems S, Dubois P, Raes M. Transfection of immortalized keratinocytes by low toxic poly(2-(dimethylamino)ethyl methacrylate)-based polymers. JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 2016; 23:739-61. [PMID: 21396176 DOI: 10.1163/092050611x559430] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Skin carcinoma are among the most spread diagnosed tumours in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based polymers, with original architecture and functionalities. We tested PDMAEMA polymers with different structures: linear, with two (DEA-PDMAEMA) or three (TEA-PDMAEMA) arms. The cytotoxicity of these polymers was assessed over a wide range of apparent M n (from 7600 to 64 600). At a N/P ratio of 7.38, cytotoxicity increases with the M n. Keratinocytes were transfected with a fluorescent oligonucleotide and then analyzed by flow cytometry. For the three architectures tested, the percentage of transfected cells and abundance of internalized oligonucleotide were closely related to the M n of the polymer. Confocal microscopy and FACS analyses showed a wide spread fine granular distribution of the oligonucleotide up to 3 days post-transfection. Then, we assessed the silencing efficiency of the polymers, targeting GFP in GFP expressing keratinocytes. The maximal silencing effect (±40%) was obtained using a DEA-PDMAEMA polymer (M n = 30 300). These results suggest that PDMAEMA-based polymers can be efficiently used to transfect immortalized keratinocytes and, thus, open new perspectives in the therapy of skin carcinoma.
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Ding J, Zhao Z, Wang C, Wang CX, Li PC, Qian C, Teng GJ. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model. Brain Res 2016; 1642:209-218. [PMID: 27038754 DOI: 10.1016/j.brainres.2016.03.045] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2015] [Revised: 02/12/2016] [Accepted: 03/28/2016] [Indexed: 01/09/2023]
Abstract
Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke.
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Affiliation(s)
- Jie Ding
- Jiangsu Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School, Southeast University, Nanjing, China
| | - Zhen Zhao
- Jiangsu Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School, Southeast University, Nanjing, China
| | - Chao Wang
- Education Ministry's Key Laboratory of Developmental Genes and Human Diseases, Institute of Life Sciences, Southeast University, Nanjing, China
| | - Cong-Xiao Wang
- Jiangsu Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School, Southeast University, Nanjing, China
| | - Pei-Cheng Li
- Department of Interventional Radiology, First Affiliated Hospital of Soochow University, Suzhou, China
| | - Cheng Qian
- Jiangsu Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School, Southeast University, Nanjing, China
| | - Gao-Jun Teng
- Jiangsu Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School, Southeast University, Nanjing, China.
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Munye MM, Tagalakis AD, Barnes JL, Brown RE, McAnulty RJ, Howe SJ, Hart SL. Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer. Sci Rep 2016; 6:23125. [PMID: 26975732 PMCID: PMC4792149 DOI: 10.1038/srep23125] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Accepted: 03/01/2016] [Indexed: 02/07/2023] Open
Abstract
Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.
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Affiliation(s)
- Mustafa M Munye
- UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, United Kingdom
| | | | - Josephine L Barnes
- UCL Respiratory Centre for Inflammation and Tissue Repair, 5 University Street, London, WC1E 6JF, United Kingdom
| | - Rachel E Brown
- UCL MRC Laboratory for Molecular Cell Biology, Gower Street, London WC1E 6BT, United Kingdom
| | - Robin J McAnulty
- UCL Respiratory Centre for Inflammation and Tissue Repair, 5 University Street, London, WC1E 6JF, United Kingdom
| | - Steven J Howe
- UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, United Kingdom
| | - Stephen L Hart
- UCL Institute of Child Health, 30 Guilford Street, London, WC1N 1EH, United Kingdom
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Bilgimol JC, Ragupathi S, Vengadassalapathy L, Senthil NS, Selvakumar K, Ganesan M, Manjunath SR. Stem cells: An eventual treatment option for heart diseases. World J Stem Cells 2015; 7:1118-1126. [PMID: 26435771 PMCID: PMC4591785 DOI: 10.4252/wjsc.v7.i8.1118] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/18/2015] [Revised: 07/04/2015] [Accepted: 08/03/2015] [Indexed: 02/06/2023] Open
Abstract
Stem cells are of global excitement for various diseases including heart diseases. It is worth to understand the mechanism or role of stem cells in the treatment of heart failure. Bone marrow derived stem cells are commonly practiced with an aim to improve the function of the heart. The majority of studies have been conducted with acute myocardial infarction and a few has been investigated with the use of stem cells for treating chronic or dilated cardiomyopathy. Heterogeneity in the treated group using stem cells has greatly emerged. Ever increasing demand for any alternative made is of at most priority for cardiomyopathy. Stem cells are of top priority with the current impact that has generated among physicians. However, meticulous selection of proper source is required since redundancy is clearly evident with the present survey. This review focuses on the methods adopted using stem cells for heart diseases and outcomes that are generated so far with an idea to determine the best therapeutic possibility in order to fulfill the present demand.
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Mignon C, Sodoyer R, Werle B. Antibiotic-free selection in biotherapeutics: now and forever. Pathogens 2015; 4:157-81. [PMID: 25854922 PMCID: PMC4493468 DOI: 10.3390/pathogens4020157] [Citation(s) in RCA: 60] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2015] [Revised: 03/09/2015] [Accepted: 03/23/2015] [Indexed: 11/16/2022] Open
Abstract
The continuously improving sophistication of molecular engineering techniques gives access to novel classes of bio-therapeutics and new challenges for their production in full respect of the strengthening regulations. Among these biologic agents are DNA based vaccines or gene therapy products and to a lesser extent genetically engineered live vaccines or delivery vehicles. The use of antibiotic-based selection, frequently associated with genetic manipulation of microorganism is currently undergoing a profound metamorphosis with the implementation and diversification of alternative selection means. This short review will present examples of alternatives to antibiotic selection and their context of application to highlight their ineluctable invasion of the bio-therapeutic world.
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Affiliation(s)
- Charlotte Mignon
- Technology Research Institute Bioaster, 317 avenue Jean-Jaurés, 69007 Lyon, France.
| | - Régis Sodoyer
- Technology Research Institute Bioaster, 317 avenue Jean-Jaurés, 69007 Lyon, France.
| | - Bettina Werle
- Technology Research Institute Bioaster, 317 avenue Jean-Jaurés, 69007 Lyon, France.
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Wong SP, Argyros O, Harbottle RP. Sustained expression from DNA vectors. ADVANCES IN GENETICS 2014; 89:113-152. [PMID: 25620010 DOI: 10.1016/bs.adgen.2014.11.002] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
DNA vectors have the potential to become powerful medical tools for treatment of human disease. The human body has, however, developed a range of defensive strategies to detect and silence foreign or misplaced DNA, which is more typically encountered during infection or chromosomal damage. A clinically relevant human gene therapy vector must overcome or avoid these protections whilst delivering sustained levels of therapeutic gene product without compromising the vitality of the recipient host. Many non-viral DNA vectors trigger these defense mechanisms and are subsequently destroyed or rendered silent. Thus, without modification or considered design, the clinical utility of a typical DNA vector is fundamentally limited due to the transient nature of its transgene expression. The development of safe and persistently expressing DNA vectors is a crucial prerequisite for its successful clinical application and subsequently remains, therefore, one of the main strategic tasks of non-viral gene therapy research. In this chapter we will describe our current understanding of the mechanisms that can destroy or silence DNA vectors and discuss strategies, which have been utilized to improve their sustenance and the level and duration of their transgene expression.
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Affiliation(s)
- Suet Ping Wong
- Leukocyte Biology Section, National Heart & Lung Institute, Imperial College London, London, UK
| | - Orestis Argyros
- Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
| | - Richard P Harbottle
- DNA Vector Research, German Cancer Research Centre (DKFZ), Heidelberg, Germany
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Rim YA, Yi H, Kim Y, Park N, Jung H, Kim J, Jung SM, Park SH, Ju JH. Self in vivo production of a synthetic biological drug CTLA4Ig using a minicircle vector. Sci Rep 2014; 4:6935. [PMID: 25374010 PMCID: PMC5381501 DOI: 10.1038/srep06935] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2014] [Accepted: 10/16/2014] [Indexed: 11/08/2022] Open
Abstract
Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin fusion protein (CTLA4Ig, abatacept) is a B7/CD28 costimulation inhibitor that can ward off the immune response by preventing the activation of naïve T cells. This therapeutic agent is administered to patients with autoimmune diseases such as rheumatoid arthritis. Its antiarthritic efficacy is satisfactory, but the limitations are the necessity for frequent injection and high cost. Minicircles can robustly express the target molecule and excrete it outside the cell as an indirect method to produce the protein of interest in vivo. We inserted the sequence of abatacept into the minicircle vector, and by successful in vivo injection the host was able to produce the synthetic protein drug. Intravenous infusion of the minicircle induced spontaneous production of CTLA4Ig in mice with collagen-induced arthritis. Self-produced CTLA4Ig significantly decreased the symptoms of arthritis. Injection of minicircle CTLA4Ig regulated Foxp3(+) T cells and Th17 cells. Parental and mock vectors did not ameliorate arthritis or modify the T cell population. We have developed a new concept of spontaneous protein drug delivery using a minicircle vector. Self in vivo production of a synthetic protein drug may be useful when biological drugs cannot be injected because of manufacturing or practical problems.
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MESH Headings
- Abatacept
- Animals
- Antirheumatic Agents/immunology
- Antirheumatic Agents/metabolism
- Antirheumatic Agents/pharmacology
- Arthritis, Experimental/genetics
- Arthritis, Experimental/immunology
- Arthritis, Experimental/pathology
- Arthritis, Experimental/therapy
- DNA, Circular/administration & dosage
- DNA, Circular/biosynthesis
- Female
- Forkhead Transcription Factors/genetics
- Forkhead Transcription Factors/immunology
- Gene Expression
- Genetic Vectors/administration & dosage
- Genetic Vectors/biosynthesis
- Immunoconjugates/immunology
- Immunoconjugates/metabolism
- Immunoconjugates/pharmacology
- Immunosuppressive Agents/immunology
- Immunosuppressive Agents/metabolism
- Immunosuppressive Agents/pharmacology
- Lymphocyte Activation/drug effects
- Mice
- Mice, Inbred DBA
- Molecular Targeted Therapy
- T-Lymphocytes, Regulatory/drug effects
- T-Lymphocytes, Regulatory/immunology
- T-Lymphocytes, Regulatory/pathology
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Affiliation(s)
- Yeri Alice Rim
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, South Korea
| | - Hyoju Yi
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, South Korea
| | - Youngkyun Kim
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, South Korea
| | - Narae Park
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, South Korea
| | - Hyerin Jung
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, South Korea
| | - Juryun Kim
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, South Korea
| | - Seung Min Jung
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, South Korea
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, Republic of Korea
| | - Sung-Hwan Park
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, Republic of Korea
| | - Ji Hyeon Ju
- CiSTEM laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, South Korea
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul. 137-701, Republic of Korea
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Ong SG, Lee WH, Huang M, Dey D, Kodo K, Sanchez-Freire V, Gold JD, Wu JC. Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer. Circulation 2014; 130:S60-9. [PMID: 25200057 DOI: 10.1161/circulationaha.113.007917] [Citation(s) in RCA: 174] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs. METHODS AND RESULTS After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes. CONCLUSIONS In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.
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Affiliation(s)
- Sang-Ging Ong
- From the Stanford Cardiovascular Institute (S.-G.O., W.H.L., M.H., D.D., K.K., V.S.-F., J.D.G., J.C.W.); and Division of Cardiology, Department of Medicine (S.-G.O., W.H.L., K.K., V.S.-F., J.C.W.), Department of Radiology (M.H., D.D., J.C.W.), and Department of Cardiothoracic Surgery (J.D.G.), Stanford University School of Medicine, CA
| | - Won Hee Lee
- From the Stanford Cardiovascular Institute (S.-G.O., W.H.L., M.H., D.D., K.K., V.S.-F., J.D.G., J.C.W.); and Division of Cardiology, Department of Medicine (S.-G.O., W.H.L., K.K., V.S.-F., J.C.W.), Department of Radiology (M.H., D.D., J.C.W.), and Department of Cardiothoracic Surgery (J.D.G.), Stanford University School of Medicine, CA
| | - Mei Huang
- From the Stanford Cardiovascular Institute (S.-G.O., W.H.L., M.H., D.D., K.K., V.S.-F., J.D.G., J.C.W.); and Division of Cardiology, Department of Medicine (S.-G.O., W.H.L., K.K., V.S.-F., J.C.W.), Department of Radiology (M.H., D.D., J.C.W.), and Department of Cardiothoracic Surgery (J.D.G.), Stanford University School of Medicine, CA
| | - Devaveena Dey
- From the Stanford Cardiovascular Institute (S.-G.O., W.H.L., M.H., D.D., K.K., V.S.-F., J.D.G., J.C.W.); and Division of Cardiology, Department of Medicine (S.-G.O., W.H.L., K.K., V.S.-F., J.C.W.), Department of Radiology (M.H., D.D., J.C.W.), and Department of Cardiothoracic Surgery (J.D.G.), Stanford University School of Medicine, CA
| | - Kazuki Kodo
- From the Stanford Cardiovascular Institute (S.-G.O., W.H.L., M.H., D.D., K.K., V.S.-F., J.D.G., J.C.W.); and Division of Cardiology, Department of Medicine (S.-G.O., W.H.L., K.K., V.S.-F., J.C.W.), Department of Radiology (M.H., D.D., J.C.W.), and Department of Cardiothoracic Surgery (J.D.G.), Stanford University School of Medicine, CA
| | - Veronica Sanchez-Freire
- From the Stanford Cardiovascular Institute (S.-G.O., W.H.L., M.H., D.D., K.K., V.S.-F., J.D.G., J.C.W.); and Division of Cardiology, Department of Medicine (S.-G.O., W.H.L., K.K., V.S.-F., J.C.W.), Department of Radiology (M.H., D.D., J.C.W.), and Department of Cardiothoracic Surgery (J.D.G.), Stanford University School of Medicine, CA
| | - Joseph D Gold
- From the Stanford Cardiovascular Institute (S.-G.O., W.H.L., M.H., D.D., K.K., V.S.-F., J.D.G., J.C.W.); and Division of Cardiology, Department of Medicine (S.-G.O., W.H.L., K.K., V.S.-F., J.C.W.), Department of Radiology (M.H., D.D., J.C.W.), and Department of Cardiothoracic Surgery (J.D.G.), Stanford University School of Medicine, CA
| | - Joseph C Wu
- From the Stanford Cardiovascular Institute (S.-G.O., W.H.L., M.H., D.D., K.K., V.S.-F., J.D.G., J.C.W.); and Division of Cardiology, Department of Medicine (S.-G.O., W.H.L., K.K., V.S.-F., J.C.W.), Department of Radiology (M.H., D.D., J.C.W.), and Department of Cardiothoracic Surgery (J.D.G.), Stanford University School of Medicine, CA.
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Zuo Y, Liao S, Xu Z, Xie J, Huang W, Yu Z. A new version of targeted minicircle producer system for EBV-positive human nasopharyngeal carcinoma. Oncol Rep 2014; 32:2564-70. [PMID: 25230680 DOI: 10.3892/or.2014.3486] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Accepted: 07/18/2014] [Indexed: 11/06/2022] Open
Abstract
Targeted gene therapy needs to be implemented for future therapies to ensure efficient activity at the site of patient primary tumors or metastases without causing intolerable side-effects. One of the elements of gene therapy is vector, which includes viral and non-viral vector. In the present study, we constructed a novel non-viral targeted gene therapeutic system by using the new minicircle (MC) producing plasmid for Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC). Molecular cloning technique was used to construct plasmids and electrophoretic analysis. Dual-luciferase reporter assay was used to evaluate the expression of luciferase. Fluorescence microscope was used to detect the expression of enhanced green fluorescence protein (EGFP). We constructed a new MC producing system pMC.BESPX-origin of plasmid replication (oriP), and demonstrated that this system could produce highly purified MC-oriP. Furthermore, our results showed that MC-oriP vector produced by the new system could mediate targeted luciferase gene expression in EBV-positive NPC cells. In addition, we verified that MC could mediate enhanced transgene expression compared with parent plasmid through EGFP transfection. The present study constructed a targeted expression vector pMC.BESPX-oriP which could carry diversified therapeutic genes for EBV-positive NPC and provides a new approach for MC-based therapies.
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Affiliation(s)
- Yufang Zuo
- Cancer Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, P.R. China
| | - Sihai Liao
- Cancer Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, P.R. China
| | - Zumin Xu
- Cancer Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, P.R. China
| | - Jierong Xie
- Cancer Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, P.R. China
| | - Wenlin Huang
- State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou, Guangdong, P.R. China
| | - Zhonghua Yu
- Cancer Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong, P.R. China
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49
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Enhanced gene disruption by programmable nucleases delivered by a minicircle vector. Gene Ther 2014; 21:921-30. [PMID: 25142139 DOI: 10.1038/gt.2014.76] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2013] [Revised: 06/18/2014] [Accepted: 07/09/2014] [Indexed: 12/21/2022]
Abstract
Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group. Taken together, our results show that gene disruption using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.
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50
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A new strategy to deliver synthetic protein drugs: self-reproducible biologics using minicircles. Sci Rep 2014; 4:5961. [PMID: 25091294 PMCID: PMC4121613 DOI: 10.1038/srep05961] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2014] [Accepted: 07/17/2014] [Indexed: 01/07/2023] Open
Abstract
Biologics are the most successful drugs used in anticytokine therapy. However, they remain partially unsuccessful because of the elevated cost of their synthesis and purification. Development of novel biologics has also been hampered by the high cost. Biologics are made of protein components; thus, theoretically, they can be produced in vivo. Here we tried to invent a novel strategy to allow the production of synthetic drugs in vivo by the host itself. The recombinant minicircles encoding etanercept or tocilizumab, which are synthesized currently by pharmaceutical companies, were injected intravenously into animal models. Self-reproduced etanercept and tocilizumab were detected in the serum of mice. Moreover, arthritis subsided in mice that were injected with minicircle vectors carrying biologics. Self-reproducible biologics need neither factory facilities for drug production nor clinical processes, such as frequent drug injection. Although this novel strategy is in its very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics.
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