Gene Therapy
Danilo D. Lasic in LIPOSOMES in GENE DELIVERY, 2019
Medicine has passed in its history through several breakthroughs, from Galen and Pasteur to the introduction of anesthesia, surgery, vaccines, antibiotics, novel imaging techniques, lasers, and remote operating devices. Direct treatment on the molecular level of diseases themselves and not their symptoms may be the next important development in human therapy. Several thousand diseases can be traced to defective or missing functional genes, and it is hoped that by delivering the appropriate gene into the appropriate cells, the mutated or missing proteins can be synthesized and the signs of the disease alleviated. In the majority of cases the mutation is in the coding region of the gene. Cells are transfected with genes which are inserted into altered viruses or inserted into plasmids. An ideal disease candidate should have been caused by a single mutation in only one gene, which should have been cloned and the mutation identified.
Dealing with the invisible
Brendan Curran in A Terrible Beauty is Born, 2020
Simple in structure, bacteria reproduce by dividing in two to generate two identical cells. Bacteria need considerably fewer genes than complex human cells in order to survive; in fact, all 3,000 genes that they possess are carried on a single circular chromosome. Some bacteria possess a second, much smaller circular chromosome consisting of genes that are useful but not essential for survival. Hunters of human genes require many millions of copies of their gene of interest but of course human genes are on huge chromosomes inside their cells — not on plasmids inside bacteria. Establishing the sequence of a cloned gene and its mutated counterpart facilitates the development of a test to discover which version of the gene is present in cells from any given individual. A series of temperature changes is used to amplify the target and the entire process of gene amplification is completed within a few hours.
Science of biotechnology – Recombinant DNA technology
Ronald P. Evens in Biotechnology, 2020
The cornerstone of biotechnology is and has been recombinant DNA technology (rDNA) over the last 40+ years from 1970s and will continue well past 2020. The basic notion in rDNA technology is manufacture of human proteins from human genes in nonhuman living systems, form of genetic engineering. As DNA is translated and transformed into m-RNA, only the exons are incorporated and united through action of spliceosome into the coding m-RNA strand. In rDNA technology, a bacterial plasmid is employed that is a circular piece of DNA that normally can be transferred between cells. Plasmids are further manipulated to maximize their function with the addition of a viral DNA promoter sequence and a viral DNA enhancer sequence, which amplify the cells’ genetic functioning. The circular plasmid DNA must be cut open to accept the human DNA using unique bacterial enzymes. Host cells in rDNA are employed to incorporate the plasmids containing the human gene and allow plasmid function and protein production.
Plasmid profile and role in virulence of salmonella enterica serovars isolated from food animals and humans in Lagos Nigeria
Published in Pathogens and Global Health, 2019
Ajayi Abraham, Smith Stella Ifeanyi, Fowora Muinah, Bode-Sojobi Ibidunni Oreoluwa, Kalpy Julien Coulibaly, Adeleye Isaac Adeyemi
Infections caused by non-typhoidal Salmonella (NTS) are common around the world, with high morbidity and mortality rates recorded annually. Salmonella serovars harbor plasmids of various sizes which may play roles in antibiotic resistance and virulence. The aim of this study was to profile and determine the role of plasmids in ciprofloxacin resistance and virulence of Salmonella serovars. Using alkaline lysis method 25 NTS serovars from food animals and humans were assayed for plasmids. Isolates ability to resist healthy human serum, bind congo red, produce hemolysin and susceptibility to ciprofloxacin before and after plasmid curing were evaluated. Mobility of plasmids was determined by conjugation. Fifteen (60%) of the 25 Salmonella serovars harbored plasmids with sizes ranging from 0.4 to 38.4 kb. S. Budapest serovars harbored 5–9 plasmids, while S. Essen and S. Mura had six plasmids each. S. Chomedey and a S. Budapest serovar were sensitive to ciprofloxacin after plasmid curing while other serovars remained resistant to ciprofloxacin after plasmid curing. All Salmonella isolates had the ability to withstand human serum before and after plasmid curing, however, some serovars lost their ability to bind congo red after plasmid curing. All Salmonella isolates that initially displayed hemolysin activity retained their ability after curing. Thirteen (86.7%) of the 15 serovars that harbored plasmids conjugatively transferred their plasmids to E. coli K-12 (DH5α). Having Salmonella serovars that harbor transferrable plasmids in the food chain can drive antibiotic resistance and enhanced virulence of otherwise less virulence strains.
Enhancement of anti-tumor effect of plasmid DNA-carrying MUC1 by the adjuvanticity of FLT3L in mouse model
Published in Immunopharmacology and Immunotoxicology, 2018
Fu-Sheng Gao, Yu-Tao Zhan, Xu-Dong Wang, Chuan Zhang
Aim: DNA vaccines have emerged as a promising strategy for cancer immunotherapy; however, their immunogenicity is weak. Fms-like tyrosine kinase 3-ligand (Flt3L) has been exploited for its ability to increase the proliferation of dendritic cells (DCs). The aim of the present study was to investigate whether co-administration of an adjuvant plasmid expressing mouse Flt3L and a DNA vaccine of the Mucin 1 (MUC1) antigen enhances immune responses. Methods: The recombinant plasmids pVAX1-MUC1 and pVAX1-Flt3L were constructed and injected into mice intramuscularly (i.m.), followed by electroporation. The humoral and cellular immune responses after immunization were examined by enzyme linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT), respectively. To evaluate the anti-tumor efficacy of the plasmids, a mouse model of MUC1-expressing tumors was established. Results: The results showed that co-administration of an adjuvant plasmid and a DNA vaccine stimulated the production of higher titers of specific antibodies and a T cell response and suppressed the growth of subcutaneous tumors expressing MUC1. Collectively, our results indicate that a plasmid expressing murine Flt3L could stimulate stronger immune responses. Conclusion: These observations emphasize the potential of Flt3L as an adjuvant for colon cancer DNA vaccines.
The increasingly anti-tumor effect of a colonic carcinoma DNA vaccine carrying HER2 by the adjuvanticity of IL-12
Published in Immunopharmacology and Immunotoxicology, 2016
Ping Gao, Chunhua Zhang, Xiaoxia Bian, Yanjun Guo, Yueguang Wei, Li Zhang, Zhaoyang Liu, Xiuying Wang, Shumin Huang
The present study aimed to determine the effect of recombinant DNA vaccine-based human epidermal growth factor receptor-2 (HER2) and Interleukin 12 (IL-12) on the development of colonic carcinoma in mice and the potential immune mechanisms involved. Recombinant plasmids pVAX1-HER2, pVAX1-IL-12 and pVAX1-HER2-IL-12 were constructed, and injected into female mice intramuscularly (i.m.) followed by an electric pulse. The humoral and cellular immune responses after immunization were examined by enzyme linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT), respectively. To evaluate the anti-tumor efficacy of the plasmids, a mouse model with a HER2-expressing tumor was designed. Mice vaccinated with the HER2-IL-12 plasmid generated the strongest inhibition efficacy on the growth of HER2-expressing tumors and prolonged mouse survival. These observations emphasized the potential of IL-12 as an adjuvant for DNA vaccines and of vaccines based on HER2 and IL-12 as a promising treatment for colonic carcinoma.
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