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Non-viral AND transfection

Cecilia M Lotufo, Maximiliano Wilda, Adrian N Giraldez, Pablo R Grigera, Nora M Mattion
A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20 residue both resulted in a 70-80% reduction in luciferase activity when compared with wild-type controls...
March 13, 2018: Archives of Virology
Wing-Fu Lai, Wing-Tak Wong
Polymeric carriers have emerged as major non-viral alternatives for gene delivery due to their lower immunogenicity and pathogenicity. However, during intracellular delivery of these carriers, multiple barriers have to be overcome or the efficiency of gene delivery will be impeded. A thorough understanding of these cellular impediments is pivotal to optimizing the efficiency of polymer-based gene delivery. This review delineates the major barriers encountered during intracellular delivery of polyplexes and discusses possible molecular designs to overcome these barriers...
March 7, 2018: Trends in Biotechnology
Jessica A Kretzmann, Cameron W Evans, Marck Norret, Pilar Blancafort, K Swaminathan Iyer
The potential impact of CRISPR/Cas9, TALE, and zinc finger technology is immense, both with respect to their use as tools for understanding the roles and functions of the genomic elements and epigenome modifications in an endogenous context and as new methods for treatment of diseases. Application of such technologies has drawn attention, however, to the prevailing lack of effective delivery methods. Promising viral and non-viral methods both currently fall short when the efficient delivery of large plasmids or multiple plasmids is required...
2018: Methods in Molecular Biology
Brynn R Olden, Yilong Cheng, Jonathan L Yu, Suzie H Pun
The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in treating multiple blood cancers has created a need for efficient methods of ex vivo gene delivery to primary human T cells for cell engineering. Here, we synthesize and evaluate a panel of cationic polymers for gene delivery to both cultured and primary human T cells. We show that a subset of comb- and sunflower-shaped pHEMA-g-pDMAEMA polymers can mediate transfection with efficiencies up to 50% in the Jurkat human T cell line with minimal concomitant toxicity (>90% viability)...
March 5, 2018: Journal of Controlled Release: Official Journal of the Controlled Release Society
Marta Holstein, Cristina Mesa-Nuñez, Csaba Miskey, Elena Almarza, Valentina Poletti, Marco Schmeer, Esther Grueso, Juan Carlos Ordóñez Flores, Dennis Kobelt, Wolfgang Walther, Manish K Aneja, Johannes Geiger, Halvard B Bonig, Zsuzsanna Izsvák, Martin Schleef, Carsten Rudolph, Fulvio Mavilio, Juan A Bueren, Guillermo Guenechea, Zoltán Ivics
The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size...
January 31, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Jieru Qiu, Lingdan Kong, Xueyan Cao, Aijun Li, Ping Wei, Lu Wang, Serge Mignani, Anne-Marie Caminade, Jean-Pierre Majoral, Xiangyang Shi
We describe a safe and highly effective non-viral vector system based on β-cyclodextrin (β-CD)-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for improved delivery small interfering RNA (siRNA) to glioblastoma cells. In our approach, we utilized amine-terminated generation 5 poly(amidoamine) dendrimers partially grafted with β-CD as a nanoreactor to entrap Au NPs. The acquired β-CD-modified Au DENPs (Au DENPs-β-CD) were complexed with two different types of therapeutic siRNA (B-cell lymphoma/leukemia-2 (Bcl-2) siRNA and vascular endothelial growth factor (VEGF) siRNA)...
February 27, 2018: Nanomaterials
Bizhan Malaekeh-Nikouei, Leila Gholami, Fariba Asghari, Saeedeh Askarian, Saeedeh Barzegar, Mehdi Rezaee, Reza Kazemi Oskuee
Non-viral vectors such as polymers and liposomes have been used as gene delivery systems to overcome intrinsic problems of viral vectors, but transfection efficiency of these vectors is lower than viral vectors. In the present study, we tried to design non-viral gene delivery vectors that mimic the viral vectors using the benefits of both cationic liposomes and cationic polymer vectors along with targeting glucocorticoid receptors to enhance cellular trafficking of vectors. Cationic liposomes containing DOTAP and cholesterol were prepared by thin-film hydration following extrusion method...
February 21, 2018: Colloids and Surfaces. B, Biointerfaces
Najla Altwaijry, Sukrut Somani, John A Parkinson, Rothwelle J Tate, Patricia Keating, Monika Warzecha, Graeme R Mackenzie, Hing Y Leung, Christine Dufès
The possibility of using gene therapy for the treatment of prostate cancer is limited by the lack of intravenously administered delivery systems able to safely and selectively deliver therapeutic genes to tumors. Given that lactoferrin (Lf) receptors are overexpressed on prostate cancer cells, we hypothesized that the conjugation of Lf to generation 3-diaminobutyric polypropylenimine dendrimer would improve its transfection and therapeutic efficacy in prostate cancer cells. In this study, we demonstrated that the intravenous administration of Lf-bearing DAB dendriplexes encoding TNFα resulted in the complete suppression of 70% of PC-3 and 50% of DU145 tumors over one month...
November 2018: Drug Delivery
Kelei Guo, Xian Lin, Yongtao Li, Wei Qian, Zhong Zou, Huanchun Chen, Hongbo Zhou, Meilin Jin
Non-structural 1 (NS1) protein is a key virulence factor that regulates replication of influenza virus. A recombinant H5N1 virus lacking the eIF4GI-binding domain of NS1 (rNS1-SD30) exhibits significantly lower pathogenicity than H5N1 virus with an intact eIF4GI-binding domain (rNS1-wt). To further investigate this phenomenon, we performed comparative proteomics analyses to profile host proteins in chicken embryo fibroblasts (CEFs) infected with rNS1-wt and rNS1-SD30 viruses. In total, 81 differentially expressed (DE) proteins were identified at 12, 24, and 36 h post-infection...
February 2, 2018: Oncotarget
Natalia Becerra, Claudia Marcela Arenas Gómez, Maria Isabel Patiño, Jean Paul Delgado, Carlos Enrique Muskus, Luz Marina Restrepo
Non-viral transfection has important implications on gene therapy, because of its safety. In particular, polyfection and nucleofection are two widely used systems for non-viral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid which carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblast and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (Human Cathelicidin Antimicrobial Peptide) and two reporter genes (Red or Green Fluorescent Protein) that included or not an Internal Ribosome Entry Site (IRES)...
February 28, 2018: Tissue Engineering. Part C, Methods
Hassan Elsana, Svetlana Mysina, Emal Ali Elkordy, Jane Carr-Wilkinson, Amal Ali Elkordy
Gene therapy is an expanding field and it can treat genetic and acquired diseases. It was found that formulations with DNA: CM-β-CD (Carboxymethyl-beta-cyclodextrin): Pluronic-F127 1:3:3 and 1:3 DNA: CM-β-CD are the most stable formulations indicating high incorporation of DNA within CM-β -CD. Gel electrophoresis revealed DNA with low CM-β -CD concentration has formed a more stable complex. Samples 1:3 DNA: CM-β-CD and 1:3:3 DNA: CM-β-CD: Pluronic-127 show no DNA fragment suggesting good condensation of DNA inside cyclodextrin cavity...
February 25, 2018: Current Drug Delivery
Erik V Munsell, Deepa S Kurpad, Theresa A Freeman, Millicent O Sullivan
Skeletal tissue regeneration following traumatic injury involves a complex cascade of growth factor signals that direct the differentiation of mesenchymal stem cells (MSCs) within the fracture. The necessity for controlled and localized expression of these factors has highlighted the role gene therapy may play as a promising treatment option for bone repair. However, the design of nanocarrier systems that negotiate efficient intracellular trafficking and nuclear delivery represents a significant challenge. Recent investigations have highlighted the roles histone tail sequences play in directing nuclear delivery and activating DNA transcription...
February 23, 2018: Acta Biomaterialia
Zhiying Wang, He Shen, Saijie Song, Liming Zhang, Wei Chen, Jianwu Dai, Zhijun Zhang
Delivery of functional genes into stem cells shows great application prospect in DNA-based tissue engineering. However, comparing with epithelial cells and cancer cells, stem cells usually exhibit low gene transfection efficiency. To enhance the transfection efficiency, non-viral gene delivery in combination with biomaterial scaffolds, has raised increasing interests from researchers in tissue engineering. Nanofibers fabricated by electrospinning technique mimicking extracellular matrix (ECM) are widely used in tissue engineering applications...
April 1, 2018: Journal of Nanoscience and Nanotechnology
Jude Akinyelu, Moganavelli Singh
The biodegradable polymer, poly(lactide-co-glycolide) is a popular polymer of choice in many nanotherapeutic studies. Herein, we report on the synthesis and evaluation of four chitosan stabilized poly(lactide-co-glycolide) nanoparticles with and without coating with gold, and the targeting ligand, folic acid, as potential non-viral gene delivery vectors. The poly(lactide-co-glycolide) nanoparticles were synthesized via nanoprecipitation/solvent evaporation method in conjunction with the surface functionalizing folic acid and chitosan...
July 1, 2018: Journal of Nanoscience and Nanotechnology
Brian T Grisez, Justin J Ray, Phillip A Bostian, Justin E Markel, Brock A Lindsey
Osteosarcoma is rare and little improvement in survival rates has occurred in the last 25 years despite modern chemotherapeutic treatment. Bioluminescent cell lines for the modeling of osteosarcoma have shown success in tracking metastases in vivo, but commonly use adenoviral vectors to transfect the native cell line with bioluminescent reporters. The purpose of this study was to develop an orthotopic model for metastatic osteosarcoma capable of in vivo monitoring of metastatic and primary tumor burden in an immunocompetent mouse and compare that model to its wild type pathogenesis...
February 10, 2018: Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society
Pratheppa Rajagopal, Sowmiya Duraiswamy, Swaminathan Sethuraman, Giridhara R Jayandharan, Uma Maheswari Krishnan
AIM: The advantages and critical aspects of nano-dimensional polymer-coated viral vector systems potentially applicable for gene delivery are reviewed in this article. DISCUSSION: Various viral and non-viral vectors have been explored for gene therapy. Viral gene transfer methods though highly efficient, are limited by their immunogenicity. Non-viral vectors have lower transfection efficiency due to their inability to escape from the endosome. To overcome these drawbacks, novel nanotechnology-mediated interventions that involve coating or modification of virus using polymers have emerged as a new paradigm in gene therapy...
February 8, 2018: Journal of Gene Medicine
Qian Qian Yang, Yi Xiang Shao, Lu Zhong Zhang, You Lang Zhou
Tendon injuries are a common injury of musculocutaneous system. Due to the lack of sufficient cellularity and low growth factor activity, healing of disrupted digital flexor tendon is troublesome and the process is lengthy and ineffective. bFGF and VEGFA gene were proved to be responsible and critical for promoting tendon healing. How to continuously enhance expression of these genes is a challenge. In this study, we developed a combination therapeutic approach that corrects the fundamental problem underlying intrasynovial tendon healing with introduction of growth factor genes via non-viral vector nanoparticle...
January 27, 2018: Colloids and Surfaces. B, Biointerfaces
Daniele Maiolo, Jessica Colombo, Jennifer Beretta, Chiara Malloggi, Gabriele Candiani, Francesca Baldelli Bombelli
Polyplexes (PX) are soft materials, obtained by blending polycations and nucleic acids, designed for gene delivery applications. While much is known about the transfection properties of PX, their protein corona, the biomolecules interacting with colloids once in a biological environment, represents an underlooked parameter in gene transfection. In this study, linear and branched polyethylenimines (lPEI and bPEI), the golden standard among non-viral vectors, were selected and used throughout the work: their physicochemical properties and protein corona when complexed to DNA were studied and linked to the toxicity and transfection efficiency arisen upon their delivery to cells...
January 30, 2018: Colloids and Surfaces. B, Biointerfaces
Marek Bednarczyk, Izabela Kozłowska, Paweł Łakota, Agata Szczerba, Katarzyna Stadnicka, Takashi Kuwana
Transgenic chickens have, in general, been produced by two different procedures. The first procedure is based on viral transfection systems. The second procedure, the non-viral method, is based on genetically modified embryonic cells transferred directly into the recipient embryo. In this review, we analyzed the effectiveness of important elements of the non-viral, cell-based strategy of transgenic chicken production. The main elements of this strategy are: isolation and cultivation of donor embryonic cells; transgene construction; cell transfection in vitro; and chimera production: injection of cells into recipient embryos, raising and identification of germline chimeras, mating germline chimeras, transgene inheritance, and transgene expression...
January 25, 2018: Journal of Applied Genetics
Daniela Benati, Fabienne Cocchiarella, Alessandra Recchia
The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a "cut and paste" SB mechanism...
January 12, 2018: Journal of Visualized Experiments: JoVE
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