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Order Blubervirales: Core Protein
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Third, the highly ingenious SplitCore technology was elaborated by the Michael Nassal’s team (Skamel et al. 2006; Nassal et al. 2008; Walker et al. 2008, 2011; Kolb et al. 2015a; Heger-Stevic et al. 2018). The SplitCore was based on the ability of two parts of the HBc molecule, coreN and coreC, to efficiently form correct HBc VLPs during expression in E. coli. The SplitCore tolerated numerous long fusions (more than 300 aa) to coreN and/or coreC and paved the way to the generation of complex triple-layered VLPs (Walker et al. 2011; Lange et al. 2014; Kolb et al. 2015a, b). The highly attractive SplitCore idea inspired other authors to present arrays of the receptor-contacting epitopes of human IgE on the surface of HBc VLPs (Baltabekova et al. 2015). The SplitCore methodology was used further to elaborate an intein-mediated trans-splicing technique, where split inteinC (intC) was added to the C-terminus of the split HBc N-core (Wang Z et al. 2021). While the split HBc with the insertion of intC at the C-terminus of N-core (designated as HBc N-intC-C) existed in inclusion bodies, the introduction of a soluble tag, gb1, to the intC C-terminus remarkably improved the solubility of recombinant protein (named HBc N-intC-gb1-C). The newly designed recombinant spontaneously assembled into the VLPs and were endowed efficiently, coupling two different model antigens onto HBc N-intC-gb1-C VLPs. The model antigens delivered by the intein-driven HBc VLP scaffold induced a dramatically enhanced antigen-specific immune response (Wang Z et al. 2021).
Companion Animals Models of Human Disease
Published in Rebecca A. Krimins, Learning from Disease in Pets, 2020
other hand, whereas all homozygotes developed diastolic dysfunction, few heterozygotes developed minor regional myocardial diastolic dysfunction without LVH, suggesting that diastolic dysfunction could be the first feature of the disease, such as observed in heterozygous human patients and mouse model of HCM. Importantly, the c.91G>C mutation results in a lower amount of cMyBP-C protein in the heart in both heterozygous and homozygous Maine Coon cats, such as seen in human HCM. This suggests regulation of mutation expression by protein quality control mechanisms, such as the ubiquitin–protein system, which has been shown to be involved after MYBPC3 gene transfer in cardiac myocytes and in vivo in the Mybpc3-targeted knock-in mice. Therefore, cats with HCM represent a good intermediary model between the numerous induced mouse models and human disease states to evaluate different causal therapeutic strategies to prevent the development of heart failure and/or sudden cardiac death or to rescue the phenotype in both heterozygotes and homozygotes for MYBPC3 mutations. Recent evidence that RNA-based therapies, such as exon skipping or trans-splicing, can repair Mybcp3 mRNA, and more recently, that Mybpc3 gene therapy long term prevents the development of the disease phenotype in Mybpc3-targeted knock-in mice, paved the way to evaluate these strategies in cats.
Imaging Cellular Networks and Protein-Protein Interactions In Vivo
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Snehal Naik, Britney L. Moss, David Piwnica-Worms, Andrea Pichler-Wallace
The salient limitations of the split-intein-based luciferase complementation systems arise from the self-catalytic nature of the DnaE gene, which, while forming the basis of the trans splicing, also results in very high-background luminescence, purportedly due to the splicing event occurring even when there is partial association of the DnaE fragments (23). In addition, reconstitution of spliced luciferase is permanent, providing only an “on” signal. Even a fleeting interaction of the proteins of interest will result in luminescence signal and furthermore, disassociation of the protein complexes of interest occurring subsequent to their association cannot be monitored. Thus, while certain initial rates of kinetic reactions could be estimated, quantitative titration curves of protein- and drug-mediated equilibrium reactions cannot be accurately measured. Finally, the truly important aspects of studying protein-protein interactions in living cells or animals lie in the ability to do so in real time. The inevitable delay in the ability to detect an interaction using this strategy can be attributed to the time required for the splicing reaction. While this may not be a factor for slow reactions occurring over long time frames, numerous drugs, chemicals and natural ligands exert their effects in seconds to minutes, and the split-intein strategy precludes the study of these important protein-protein interactions in cells and live animals.
Nucleic acid therapeutics: a focus on the development of aptamers
Published in Expert Opinion on Drug Discovery, 2021
Swati Jain, Jaskirat Kaur, Shivcharan Prasad, Ipsita Roy
Small nucleic acids may also act via trans-splicing at the post-transcriptional level. Trans-splicing is catalyzed by the cellular spliceosome machinery which substitutes intrinsic unwanted sequences in the pre-mRNA with the correct sequence containing built-in donor and acceptor splice sites and a hybridization domain for targeted action. This results in a mutation-free, chimeric functional mRNA transcript [15–17]. Spliceosome-mediated RNA trans-splicing (SMaRT) was used to correct LMNA (lamin A)-related congenital muscular dystrophy in mice [16]. Pre-trans-spliced molecules (PTMs) containing the wild-type lamin A coding sequence, flanked by splicing sites and a hybridization domain were used to target mutant intron 5 of lamin A pre-mRNA by an adenoviral vector. Although the trans-splicing efficiency was quite low, it was sufficient to improve phenotype in homozygous LmnaΔK32 mice [16], showing the promise of the approach.
Virus-associated ribozymes and nano carriers against COVID-19
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2021
Beyza Dönmüş, Sinan Ünal, Fatma Ceren Kirmizitaş, Nelisa Türkoğlu Laçin
Hammerhead ribozymes catalyse cleavage reactions through the Watson–Crick base pairing upon target mRNA molecules. Thus, gene expression can be down-regulated. Ribozymes can be targeted against dominant mutations due to flexible targeting and arrangement properties on their arms [103]. Mutant protein production causes some genetic diseases. Therefore, gene expression can be down-regulated by ribozymes against the mutant transcript due to specific base-pairing properties of ribozymes to target genes [104]. Although ribozymes interfere gene expression, the RNA trans-splicing process enables them to repair the mutant RNAs. The process demonstrates the genetic information can be revised. The mutant-RNA repair mechanism performed by trans-splicing group I intron ribozymes, and includes the replacement of the wild-type sequence to the wild type designed to be carried in the ribozyme. [105]. Group I intron ribozyme mediated RNA-trans splicing method is an alternative gene therapy for the treatment of genetic disorders with revision without eliminating mutant transcript permanently [106]. Even though strong evidence exists about the method as usable, there are some disadvantages such as the short length of the ribozyme binding region and low sequence specificity. Ribozymes designed to use against viral infections targeting viral RNA molecules are a potential anti-viral agent. For instance, studies have shown the inhibition of viral gene replication and expression in various cell lines by the hammerhead and hairpin ribozymes designed against the human immunodeficiency virus known as HIV [107].
Exploiting differential RNA splicing patterns: a potential new group of therapeutic targets in cancer
Published in Expert Opinion on Therapeutic Targets, 2018
Nidhi Jyotsana, Michael Heuser
Trans-splicing, also known as spliceosome-mediated RNA trans-splicing (SMART) replaces the entire target splice site sequence (5ʹ or 3ʹ). A plasmid is used to express an ASO targeting the mutated splice site, a synthetic splice site that directs splicing, and a desired copy of RNA sequence that will be spliced instead of the mutated one to restore the correct mRNA expression [141]. SMART has been used to correct factor VIII deficiency in hemophilia mice [142]. Also, ribozymes that use trans-splicing have been introduced to correct defective p53 and beta-globin mRNA in vitro in human cancer cells and erythrocyte precursors, respectively [143,144]. Viral delivery of ribozyme for specific trans-splicing in hTERT expressing cells sensitized them to prodrug treatment and reduced tumor progression in a peritoneal carcinomatosis nude mouse model [145]. Limited in vivo delivery and use of expression vectors is a challenge for trans-splicing. Targeting of all tumor cells is therefore unlikely to be achieved with this technology and makes this technique useful when only a small increase in therapeutic RNA in a minor population of cells is sufficient.