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Adenoviral Vectors for Gene Therapy of Inherited and Acquired Disorders of the Lung
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
David T. Curiel, Robert I. Garver
The most widely used adenoviral vectors contain a deletion within the El region that inactivates both of the two regions, E1A and E1B. The complex biology of these two genes and protein products have been comprehensively reviewed (see 4-6), but key features relevant to adenoviral vectors will be briefly described here. The two El regions have separate transcriptional regulatory signals, and each region actually codes for multiple proteins generated by differential splicing of the transcripts. The function of the three major E1A proteins are multifaceted and complex, but for the purpose of this discussion, the most relevant activity is the promiscuous transactivation of viral gene promoters by one of the E1A proteins. This transactivation is considered a primary initiating event of viral replication, and in its absence, viral replication occurs at negligible levels except under very specific conditions, such as a high multiplicity of infection (7-10). The E1A proteins by themselves appear to be strong inducers of apoptosis (11-13). In the context of adenoviral replication, one of the most important functions of the E1B proteins is to prevent the onset of apoptosis in the presence of E1A (12,14). Both the E1A and E1B proteins can be supplied in trans to replication-enable El-defective adenoviruses. This has been most accomplished by the use of the 293 cell line, which is human embryonic kidney cells containing the E1A and E1B gene region of human adenovirus serotype 5 (3,15).
Genetics and Mutants
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
The amber suppressor genes restricted formation of infective centers, while each infected Su+ host, once the infection was established, produced a normal phage yield. The number of infective centers formed by infection of the Su+ cells by azure mutants depended on the multiplicity of infection. For infection of Su-3 cells with an azure mutant, the response was a linear function of multiplicity of infection up to multiplicities of about 10, suggesting that the infective centers formed by Su+ strains were not due to heterogeneity in the population of the cells; i.e., when Su+ cells were infected with azure mutants, there was a certain probability of forming an infective center per phage particle, not per cell, and this probability depended on the particular Su+ gene present. Attempts to rescue or reverse the abortive infection by simultaneous infection with wild-type or amber mutants of f2 have failed.
Chikungunya Virus Infection
Published in Venkatesan Jayaprakash, Daniele Castagnolo, Yusuf Özkay, Medicinal Chemistry of Neglected and Tropical Diseases, 2019
D. Velmurugan, K. Manish, D. Gayathri
Das et al. (2016) designed and validated few inhibitors against CHIKV. It has been reported that compounds 1, 2, 3, and 4 (Figure 5) suppress CHIKV RNA synthesis and infectious virus release. Among them, compound 4 was reported to be a very efficient inhibitor of viral RNA synthesis which was clearly translated into prominent suppression of progeny virus release. Compounds 5 and 3 were shown to be more potent inhibitors of virus replication (EC50: 1.5 μM and 11 μM, respectively) than their potential to inhibit the protease activity of nsP2. Based on these results, it has been concluded that since the pronounced inhibitory effect of compound 3 was detected in experiments carried out using low MOI (multiplicity of infection) (virus inhibition) but not using a high MOI (positive-strand RNA synthesis), compound 3 might inhibit virus spread in cell culture which is crucial under low-MOI.
Development and Clinical Translation Considerations for the Next Wave of Gene Modified Hematopoietic Stem and Progenitor Cells Therapies
Published in Expert Opinion on Biological Therapy, 2022
Matthew Li, Brent Morse, Sadik Kassim
As noted in previous sections, starting materials play a critical role in the success of CGT manufacturing processes, and therefore maintaining a consistent level of quality for these materials is essential. In some cases, e.g. off the shelf reagents that are commonly used in CGT manufacture, the control may simply consist of confirming that the material’s certificate of analysis meets the specification. For custom materials, or reagents that may have a greater impact on the product quality, a significant amount of custom assay development may be required. For viral vectors, this may include custom assays to establish the titer and the multiplicity of infection (MOI) for each batch, whereas gene editing reagents such as enzymes, single guide RNA, or mRNA, may require development of many orthogonal assays to fully characterize their potency.
Isolation, characterization, and application of Salmonella paratyphi phage KM16 against Salmonella paratyphi biofilm
Published in Biofouling, 2021
Liming Jiang, Rui Zheng, Qiangming Sun, Chenghua Li
Phage KM16 had the highest activity after treatment for 1h at 42°C, which noticeably declined at 50°C and was completely inactivated at 90°C (Figure 2a). The results showed that phage KM16 has a low temperature adaptability, which is consistent with the optimum survival temperature of S. paratyphi A NA3. Phage KM16 produced the most plaques at pH = 6 ∼ 7, and plaque production was significantly higher at pH = 10 ∼ 11 than at pH = 3 ∼ 4 (Figure 2b). These results indicate that phage KM16 had good tolerance to alkaline conditions but was extremely intolerant to acidic conditions. The multiplicity of infection (MOI) refers to the ratio of the number of phage to bacteria. The optimum MOI of phage KM16 was 0.0001; the plaque of phage KM16 decreased significantly at an MOI of 0.001 and reached a minimum at an MOI of 100 (Figure 2c).
Influenza A virus infection induces indoleamine 2,3-dioxygenase (IDO) expression and modulates subsequent inflammatory mediators in nasal epithelial cells
Published in Acta Oto-Laryngologica, 2020
Yi-Tsen Lin, Chih-Feng Lin, Te-Huei Yeh
All segmented expression plasmids of influenza A Virus (IAV) were kindly provided by Dr. Shin-Ru Shih of Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taiwan. Mouse adapted PR8 and recombinant IAV of mouse adapted PR8 were generated using a reverse genetics system according to previous reports [6]. Briefly, 293 T cells were transfected using 15 μL Trans IT-LT1 (Mirus Bio LLC) with 1 μg per plasmid. Recombinant IAV were harvested and propagated in 10 day-old embryonated chicken eggs. Harvested viruses were aliquoted and stored at −80 °C until further use. For IAV infection using influenza PR8 virus, the multiplicity of infection (moi):10 is applied to infect the epithelial cells prepared as above. When required, the tryptophan and/or indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyltryptophan (1-MT) was added at a concentration of 0.2 mM for pre-treatment and re-added in 24 hrs.