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Archives de Pediatrie : Organe Officiel... Feb 2020Pathogenic variants of the CFTR gene are responsible for a broad phenotypic spectrum characterized by malfunction of some exocrine tissues, with an autosomal recessive... (Review)
Review
Pathogenic variants of the CFTR gene are responsible for a broad phenotypic spectrum characterized by malfunction of some exocrine tissues, with an autosomal recessive mode of inheritance. More than 2,000 variants, distributed throughout the CFTR gene, have been identified, with different effects on the gene and protein expression and function. Genotype-phenotype correlation studies have associated severe variants with a typical multi-organ form of cystic fibrosis, while mild variants are involved in monosymptomatic or adult-onset diseases, called CFTR-related disorders. However, the interpretation of rare variants remains challenging. This review presents an overview of the epidemiology of CFTR variants worldwide and in France and describes the functional classification. Finally, some frequent cystic fibrosis-causing and mild CFTR variants are used as example to depict the molecular pathology of the CFTR locus. Finally, we give the recommendations concerning nomenclature and classification that are useful for appropriate genetic counseling. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.
Topics: Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Exons; Gene Rearrangement; Humans; Inteins; Mutation; Phenotype; RNA, Untranslated
PubMed: 32172939
DOI: 10.1016/S0929-693X(20)30044-0 -
Science Translational Medicine May 2019Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest...
Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.
Topics: Animals; Dependovirus; Gene Transfer Techniques; Genetic Vectors; Green Fluorescent Proteins; Humans; Induced Pluripotent Stem Cells; Inteins; Mice; Organoids; Phenotype; Photoreceptor Cells, Vertebrate; Retina; Swine; Trans-Splicing
PubMed: 31092694
DOI: 10.1126/scitranslmed.aav4523 -
Nucleic Acids Research Jul 2015Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb),...
Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; Cell Line; Deoxyribonucleases; Dependovirus; Gene Targeting; Genetic Therapy; Humans; Inteins; Plasmids; Streptococcus pyogenes; Transfection
PubMed: 26082496
DOI: 10.1093/nar/gkv601 -
Nature Apr 2023Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in...
Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.
Topics: Humans; Cell Nucleus; Chromatin; Cross-Linking Reagents; Energy Transfer; Epigenomics; Inteins; Iridium; Mutation; Neoplasms; Photosensitizing Agents; Protein Binding; Protein Interaction Maps
PubMed: 37020029
DOI: 10.1038/s41586-023-05914-y -
Genes & Development Oct 2014Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor...
Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA.
Topics: Base Pairing; Base Sequence; Humans; Inteins; Intracellular Signaling Peptides and Proteins; Microsatellite Repeats; Molecular Sequence Data; Mutation; Plasmids; Protein Serine-Threonine Kinases; RNA; RNA Splice Sites; RNA, Circular; Transcription Factors
PubMed: 25281217
DOI: 10.1101/gad.251926.114 -
Current Biology : CB Mar 2017Lennon and Belfort introduce inteins - protein introns - and describe how they escape host proteins, their uses in biotechnology, where they are found in nature, and...
Lennon and Belfort introduce inteins - protein introns - and describe how they escape host proteins, their uses in biotechnology, where they are found in nature, and their role in post-translational regulation.
Topics: Animals; Biotechnology; Exteins; Humans; Inteins
PubMed: 28324730
DOI: 10.1016/j.cub.2017.01.016 -
Biosensors Apr 2022Cell-based sensors provide a flexible platform for screening biologically active targets and for monitoring their interactions in live cells. Their applicability extends... (Review)
Review
Cell-based sensors provide a flexible platform for screening biologically active targets and for monitoring their interactions in live cells. Their applicability extends across a vast array of biological research and clinical applications. Particularly, cell-based sensors are becoming a potent tool in drug discovery and cell-signaling studies by allowing function-based screening of targets in biologically relevant environments and enabling the in vivo visualization of cellular signals in real-time with an outstanding spatiotemporal resolution. In this review, we aim to provide a clear view of current cell-based sensor technologies, their limitations, and how the recent improvements were using intein-mediated protein engineering. We first discuss the characteristics of cell-based sensors and present several representative examples with a focus on their design strategies, which differentiate cell-based sensors from in vitro analytical biosensors. We then describe the application of intein-mediated protein engineering technology for cell-based sensor fabrication. Finally, we explain the characteristics of intein-mediated reactions and present examples of how the intein-mediated reactions are used to improve existing methods and develop new approaches in sensor cell fabrication to address the limitations of current technologies.
Topics: Biosensing Techniques; Inteins; Protein Engineering; Signal Transduction
PubMed: 35624584
DOI: 10.3390/bios12050283 -
Microorganisms Dec 2020Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing.... (Review)
Review
Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing. Since their discovery in the 1990s, recent advances in intein technology allow for them to be implemented as a modern biotechnological contrivance. Radical improvement in the structure and catalytic framework of - and -splicing inteins devised the development of engineered inteins that contribute to various efficient downstream techniques. Previous literature indicates that implementation of intein-mediated splicing has been extended to in vivo systems. Besides, the homing endonuclease domain also acts as a versatile biotechnological tool involving genetic manipulation and control of monogenic diseases. This review orients the understanding of inteins by sequentially studying the distribution and evolution pattern of intein, thereby highlighting a role in genetic mobility. Further, we include an in-depth summary of specific applications branching from protein purification using self-cleaving tags to protein modification, post-translational processing and labelling, followed by the development of intein-based biosensors. These engineered inteins offer a disruptive approach towards research avenues like biomaterial construction, metabolic engineering and synthetic biology. Therefore, this linear perspective allows for a more comprehensive understanding of intein function and its diverse applications.
PubMed: 33339089
DOI: 10.3390/microorganisms8122004 -
Frontiers in Molecular Biosciences 2022Multidrug-resistant pathogens are of significant concern in recent years. Hence new antifungal and anti-bacterial drug targets are urgently needed before the situation... (Review)
Review
Multidrug-resistant pathogens are of significant concern in recent years. Hence new antifungal and anti-bacterial drug targets are urgently needed before the situation goes beyond control. Inteins are polypeptides that self-splice from exteins without the need for cofactors or external energy, resulting in joining of extein fragments. Inteins are present in many organisms, including human pathogens such as , , , and . Because intein elements are not present in human genes, they are attractive drug targets to develop antifungals and antibiotics. Thus far, a few inhibitors of intein splicing have been reported. Metal-ions such as Zn and Cu, and platinum-containing compound cisplatin inhibit intein splicing in and by binding to the active site cysteines. A small-molecule inhibitor 6G-318S and its derivative 6G-319S are found to inhibit intein splicing in and with a MIC in nanomolar concentrations. Inteins have also been used in many other applications. Intein can be used in activating a protein inside a cell using small molecules. Moreover, split intein can be used to deliver large genes in experimental gene therapy and to kill selected species in a mixed population of microbes by taking advantage of the toxin-antitoxin system. Furthermore, split inteins are used in synthesizing cyclic peptides and in developing cell culture model to study infectious viruses including SARS-CoV-2 in the biosafety level (BSL) 2 facility. This mini-review discusses the recent research developments of inteins in drug discovery and therapeutic research.
PubMed: 35211511
DOI: 10.3389/fmolb.2022.821146 -
Nature Communications Oct 2019Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here...
Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.
Topics: CRISPR-Cas Systems; Cell Line, Tumor; Cinnamates; Drug Resistance, Bacterial; Gene Editing; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; HEK293 Cells; HeLa Cells; Humans; Hygromycin B; Induced Pluripotent Stem Cells; Inteins; Lentivirus; Luminescent Proteins; Neomycin; Nucleosides; Protein Splicing; Puromycin; Trans-Splicing; Transgenes
PubMed: 31672965
DOI: 10.1038/s41467-019-12891-2