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Toward Correction of the Genetic Defect in Cystic Fibrosis
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
Larry G. Johnson, Richard C. Boucher
Efficient cationic liposome-mediated gene transfer has been reported in vitro in a variety of cell types (85-89) and in the alveolar region of the lung in vivo (90). However, the efficiency of cationic liposome-mediated gene transfer to well differentiated airway epithelia in vivo is low and patchy. Studies designed to identify rate limiting factors for liposome-mediated gene transfer in undifferentiated cell lines in vitro suggest that nuclear (but not cellular) entry is rate-limiting in nontransfectable undifferentiated cells (91). In contrast, gene transfer to well-differentiated airway epithelial cells is limited by failure of DNA-liposome complexes to enter the cell, preventing efficient gene transfer (55). This inability to enter differentiated airway epithelial cells arises from a loss of phagocytic entry mechanisms in differentiated columnar airway epithelial cells (55). The issue of repetitive dosing with this transient expression vector system has not been addressed in detail.
Gene Therapy
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
Currently, in addition to efficient and safe gene delivery, the duration of expression, especially in the case of nonviral vectors, seems to be the largest unsolved problem. In principle, depending on the disease, a short-term transient expression or a long-term sustained expression can be designed by using appropriate DNA cassettes. DNA sequences can regulate rate, duration, and location of expression. While current nonviral delivery systems aim for episomal delivery (i.e., not integrating into the chromosome), the next generation of plasmids may contain sequences for adhesion to the nucleus and self-replication. They may also contain peptide stretches specifying nuclear localization (Boulikas, 1995; 1996), site-specific chromosome integration, and possibly homologous recombination.
Gene Therapy
Published in John C Watkinson, Raymond W Clarke, Louise Jayne Clark, Adam J Donne, R James A England, Hisham M Mehanna, Gerald William McGarry, Sean Carrie, Basic Sciences Endocrine Surgery Rhinology, 2018
Seiji B. Shibata, Scott M. Graham
The shortcomings of viral vectors have led to the investigation of non-viral delivery systems. These have included purified or naked DNA in plasmid form or ballistic gene delivery, the so-called gene gun. Only exposed surfaces accessible to a microcarrier coated with DNA are candidates for the gene gun. Although successful in mice lungs this approach is difficult to incorporate in animals with larger lungs due to the lack of equipment design that would match the lung size. Most interest in non-viral gene delivery has centred on liposomes. Liposomes bind to DNA, spontaneously forming complexes that have high affinity for plasma cell membranes. DNA containing liposomes are incorporated into the cell by endocytosis. The advantages of non-viral vectors mainly revolve around safety. They are non-immunogenic and there is no potential for insertion mutagenesis. The main difficulties with non-viral vectors relate to transient expression and less efficient gene transfer as compared to viral vectors.
Modern vaccine strategies for emerging zoonotic viruses
Published in Expert Review of Vaccines, 2022
Atif Ahmed, Muhammad Safdar, Samran Sardar, Sahar Yousaf, Fiza Farooq, Ali Raza, Muhammad Shahid, Kausar Malik, Samia Afzal
Plants have been utilized as a platform for the synthesis of biopharmaceuticals for more than 30 years, a strategy known as molecular farming, which has numerous benefits over alternative expression systems, including economics, scalability, and safety. When transient expression systems are utilized, the most important benefit is that they can be ramped up quickly to meet unexpected and unforeseen demands, making them an excellent platform for vaccine manufacturing in pandemics like the one we are presently experiencing. Several molecular farming firms specializing in the creation of plant-derived proteins attest to the effectiveness of this strategy like Medicago Inc. (Quebec City, QC, Canada) and Agrenvec (Madrid, Spain). Both the strategies to use subunit protein or VLP as a vaccine have been studied extensively in plants against SARS Cov-2. Several vaccines have also been obtained using this technique. The creation of VLPs as an alternative to subunit vaccinations offers several advantages, including the ability to elicit even greater cellular and humoral responses due to the ordered antigen organization. Medicago Inc. (Quebec City, QC, Canada) disclosed the synthesis of VLPs against SARS-CoV-2 in tobacco plants using a transient expression technique. This business estimates that 10 million doses of VLP-based vaccinations can be produced every month.
Hematological Characteristics of β-Globin Gene Mutation –50 (G>A) (HBB: c.-100G>A) Carriers in Mainland China
Published in Hemoglobin, 2020
Yuan Zhao, Fan Jiang, Dong-Zhi Li
The silent β-thal patients are characterized by normal hematological findings and defined only by a mildly imbalanced α-/β-globin chain synthesis ratio. It usually results from variants of the distal CACCC box, the 5′ untranslated region (5′UTR), the polyadenylation (polyA) signal and some splicing defects [13]. Homozygosity for silent alleles produces a mild thalassemia trait. The β silent/β severe genotype leads to mild β-thal. An example of silent β-thal is −101 (C>T) (HBB: c.-151C>T), a common cause of the silent β-thal phenotype in the Southern Italian population [14,15]. However, we had no cases of homozygosity for the −50 (G>A) mutation, and had only one case of compound heterozygosity for −50 (G>A) and a common β-thal without interference from α-thal variants. Our limited data cannot determinate whether it is a silent β-thal allele or a polymorphism. More cases are needed, and especially in vitro transient expression studies of the mutant gene are warranted.
Ternary complex of plasmid DNA with NLS-Mu-Mu protein and cationic niosome for biocompatible and efficient gene delivery: a comparative study with protamine and lipofectamine
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Mohammad Hadi Nematollahi, Masoud Torkzadeh-Mahanai, Abbas Pardakhty, Hossein Ali Ebrahimi Meimand, Gholamreza Asadikaram
The transient expression efficiency of the binary and ternary vectors was evaluated in the transfection assay. The pmCherry-C1 plasmid encoding Cherry fluorescent protein was used. Transfection efficiency of the binary complexes was tested at various N/P ratios varying from 1 to 11. Maximum transfection was obtained when cells were transfected with N/P = 7 for NMM/pDNA complexes and N/P = 5 for protamine complexes (data not shown). Transfection efficiency over time were studied in HEK293T and MCF-7 cell lines. As observed in Figure 11, the percentage of transfected cells was higher with the NMM binary vectors compared to vectors containing protamine and naked plasmid. In addition, transfection efficiency of ternary vectors was higher than both binary vectors and the difference between NMM ternary efficiency was significant compared to protamine ternary (p < .001). At 48 h post-transfection, the maximum percentage of transfected cells for NMM/DNA and protamine/DNA vectors were 18 and 13%, respectively. Both values were inferior to their related ternary vectors (NMM ternary 43% and protamine ternary 32%). Representative fluorescent images of transfection efficiency in HEK293T cell line and quantified results for both cell lines have been shown in Figures 11 and 12, respectively.