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Nanotechnology in Preventive and Emergency Healthcare
Published in Bhaskar Mazumder, Subhabrata Ray, Paulami Pal, Yashwant Pathak, Nanotechnology, 2019
Nilutpal Sharma Bora, Bhaskar Mazumder, Manash Pratim Pathak, Kumud Joshi, Pronobesh Chattopadhyay
The techniques available for gene transfection include: (1) the viral (transduction) method, (2) the physical (direct micro injection) method, and (3) chemical methods. On its own, the viral method is considered to be the most superior due to its specificity and efficiency in penetrating cells. It uses the theory of functional gene insertion into a nonspecific site within the viral genome, which in turn uses the host cell’s biosynthetic apparatus to expresses the genes in vivo (Mulligan, 1993). However, there is a limit to the length of the genes that can be synthesized and there are numerous difficulties that are faced with respect to its handling and large-scale production. Moreover, disadvantages like immunogenicity also limit the use of viral methods extensively (Ledford, 2007). Physical methods are used to directly penetrate the gene into the cells by utilizing fine needle punctures, high pressure gas, or simulations of electrical impulses. The primary physical methods available for gene therapy include biolistics, electroporation, ultrasound, hydrodynamic injection, and jet injection (Gao and Huang, 1995; Naldini et al., 1996; Orio et al., 2012; Panje et al., 2012). Though they have much lesser side effects when compared with viral methods, the physical methods still suffer major disadvantages related to cellular damage, low transfection, necessity of expensive instrumentation, and difficult and labor intensive protocols and manipulations (Robinson and Pertmer, 2001).
Expression of Genes in Bacteria, Yeast, and Cultured Mammalian Cells
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
The introduction of foreign DNA or RNA into mammalian cells is called transfection when it is done by nonviral methods, and infection or transduction when it is performed with a virus. The corresponding process in bacteria and yeast is usually called transformation. Transformation was discussed in Chapter 2 for cloning strains of E. coli; in this chapter, we extend the discussion to other types of microorganisms, such as Gram-positive bacteria and yeast. We then discuss mammalian cell culture and some of the myriad of ways in which foreign genes can be introduced into mammalian cells in vitro. Some of these methods are routine and others much more difficult, requiring specialized cloning or instrumentation. The choice of method depends on the downstream application and is often key to the success of an experiment. We then address some particular transfection techniques for difficult cells: electroporation, microinjection, the “gene gun,” optical and magnetic transfection, and viral vectors.
Understanding the Technologies Involved in Gene Therapy
Published in Yashwant V. Pathak, Gene Delivery Systems, 2022
Manish P. Patel, Jayvadan K. Patel, Mukesh Patel, Govind Vyas
Gene delivery systems consists of viral vectors and non-viral vectors. Non-viral vectors are safe, economical, reproducible and have no size limit for DNA transfer through a vector (Gascón et al. 2013). Non-viral vectors in gene delivery systems allow simple and safe administration with low host immunogenicity because of no viral content, although it is to be noted that it is less efficient at implementing and sustaining gene expression of foreign nucleic acids. The non-viral vectors contain naked DNA; they have identical physical and chemical properties as the original gene. It inserts via direct injection either plasmid DNA, naked DNA or by chemical or physical means (Herweijer et al. 2003). In a viral vector, the efficiency of transfecting host cells is relatively high compared to non-viral methods. The drawbacks of viral vectors are immunogenicity and cytotoxicity. The first related fatality of gene therapy in a clinical trial was linked to the inflammatory reaction to the adenovirus viral vector. Insertional mutagenesis is another cause of concern over gene transfer vehicles; for example, abnormal chromosomal integration of viral DNA disrupts the expression of a tumor suppression gene or developed oncogene which leads to malignant transformation of the cells. Because of its reduced pathogenicity, ease of production and low cost, non-viral vectors provide more safety and advantages over viral approaches. The major advantage of non-viral vectors is biosafety. However, the applications of non-viral gene transfer have been ignored in the past because of their poor efficiency in delivery and thus low transient expression of their transgenes.
Enhanced transfection efficiency of low generation PAMAM dendrimer conjugated with the nuclear localization signal peptide derived from herpesviridae
Published in Journal of Biomaterials Science, Polymer Edition, 2021
Jeil Lee, Yong-Eun Kwon, Younjin Kim, Joon Sig Choi
Viral vectors have been widely investigated as carriers to treat diseases due to high transfection and infection efficiency in host cells. Considering that all existing gene medicines and most candidates being tested in clinical trials are viral vectors, viruses may be optimized carriers for gene therapy [4, 5]. Despite these advantages, problems with viral vectors remain: renewability as pathogenic wild type, carcinogenesis by insertion mutation, excessive immune response, and enormous production and dosing costs. Many researchers have shifted attention to nonviral vectors as alternatives to the safety concern and economic problems associated with viral vectors.
Preparation and characterization of polyamidoamine dendrimers conjugated with cholesteryl-dipeptide as gene carriers in HeLa cells
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Le Thi Thuy, Minyoung Choi, Minhyung Lee, Joon Sig Choi
Gene therapy has received considerable attention owing to its potential applications in the biomedical field. Therapeutic genes are used to treat various genetic diseases, such as neurodegenerative diseases, autoimmune diseases, and numerous cancers [1]. In cancer, transfected therapeutic genes can cause cell death or inhibit the growth of cancer cells. Various gene therapy approaches have been studied for cancer treatment, including siRNA therapy, pro-apoptotic gene therapy, anti-angiogenic gene therapy, suicide gene therapy, immunotherapy, oncolytic virotherapy, and gene-directed enzyme prodrug therapy [2–7]. In November 2017, clinical trials of gene therapy were conducted in 38 countries, involving 2,597 genes. According to the results of these trials, over 65% of the tested genes were associated with cancer [8]. Naked genetic molecules cannot be internalized efficiently by target cells because of their serum nuclease sensitivity, phagocyte ingestion, fast renal removal, poor uptake by target cells, and toxic effects arising from immune response stimulation. This limitation severely restricts the clinical application of naked DNA. The development of gene vectors has significant implications for enhancing therapeutic efficacy [9, 10]. Gene vectors are essential for transferring therapeutic genes to the target cells. There are two main classes of gene vectors: viral and non-viral. Viral vectors are well known for their high efficiency in gene transfection compared to non-viral vectors. Nevertheless, there are several drawbacks of viral vectors, such as cytotoxicity, immune response, and high cost. Therefore, non-viral vectors have gained popularity in the field of gene therapy due to their biocompatibility, surface functionality in vivo or in vitro, and low-cost production [9, 11]. Hence, designing an effective non-viral gene carrier with minimized toxicity is currently an important research topic in gene carrier technology.
Lipid-based nanocarrier mediated CRISPR/Cas9 delivery for cancer therapy
Published in Journal of Biomaterials Science, Polymer Edition, 2023
Aisha Aziz, Urushi Rehman, Afsana Sheikh, Mohammed A. S. Abourehab, Prashant Kesharwani
The transfection efficiency was seen to be higher than commercial transfecting agents. The downregulation of HuR in SAS cells by CRISPR/SLN-HPR initiated key anti-proliferation signaling pathways which were enhanced by the co-administration of Epi/Lip-HPR formulation, and their combined effect showed inhibition of SAS cells by almost 69% [86].