CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts

Coevolution of expression of prion-protein and doppel genes

Gene duplication is a major mechanism for the origin of new genes. Subsequent function divergence and acquisition of new regulatory elements is the necessary step for survival of a newly duplicated gene. However, despite of existence of several models, it remains unclear how paralogs evolve after the duplication and develop new functions. An example of evolution by gene duplication is PRNP, coding for prion protein (PrP), and its downstream duplicate, PRND, coding for doppel (Dpl). Recent studies have shown that PrP possesses neuroprotective functions while Dpl is neurotoxic and induces apoptosis. Expression of PrP and Dpl is altered in various neurodegenerative disorders, such as Alzheimer's, as well as after brain injury and during development; this suggests their possible antagonistic co-regulation by a so far unknown mechanism. The aims of the work described in this thesis are to examine the evolution of the PRNP and PRND genes using three models: frog (Xenopus laevis), marsupial opossum (Monodelphis domestica) and mouse, and to define the molecular mechanisms underlying the antagonism in the functions of their proteins. Previously unreported amphibian and marsupial PRND transcripts were cloned and characterized. Tissue-expression analysis demonstrated that in frog PRND relies primarily on regulatory DNA elements of the PRNP gene and is expressed in brain as a chimeric transcript, sharing the first two 5' UTR exons of the PRNP gene. PRND acquires its own promoter in mammals and develops new tissue specificity. Conservation of the chimeric transcript in mouse and its translation (shown by polysome binding assay) in mice embryos suggests possible functional significance in development. Transcription factors that might regulate the antagonistic relationship of PrP and Dpl were predicted by bioinformatics and comparative genomics analysis of the two genes within vertebrates. The predicted FAC1/BPTF binding site, situated next to the second non-coding exon of PRNP, was selected for experimental analysis based on the literature-deduced relationship between FAC1/BPTF, PrP, and Dpl. Reporter gene assays demonstrated upregulation of luciferase expression in constructscontaining both a Prnp conserved non-coding DNA region with the predicted FAC1/BPTF binding site and a Prnd promoter region without FAC1/BPTF binding site; the latter might be explained by an indirect effect. Binding sites for the transcription factors E2F1, CREB, AP2, USF1, and NFY cluster were predicted in the PRND proximal promoter by computational analysis. Consequently, a model of PrP/Dpl co-regulation is proposed that includes recruitment of BPTF by USF1, phosphorylation of retinoblastoma protein Rb induced by FAC1/BPTF followed by E2F1 de-repression leading to activation of the PRND promoter. Discovery and characterisation of previously undetected mouse Bptf transcript variants suggests tissue-specific modulations of this mechanism, as well as having wide implications for the functions of "BPTF" and "FAC1" not previously suspected Overall, the results presented are important for understanding of evolution by gene duplication in general. For the first time, gene development is compared across three evolutionarily distant vertebrate species: amphibians, marsupials, and mammals. The proposed mechanism for PrP and Dpl co-regulation by FAC1/BPTF provides novel insights into neuronal death

CRISPR/Cas9-generated mouse model of Duchenne muscular dystrophy recapitulating a newly identified large 430 kb deletion in the human DMD gene

Exon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD

The beam and detector of the NA62 experiment at CERN

NA62 is a fixed-target experiment at the CERN SPS dedicated to measurements of rare kaon decays. Such measurements, like the branching fraction of the K(+) → π(+) ν bar nu decay, have the potential to bring significant insights into new physics processes when comparison is made with precise theoretical predictions. For this purpose, innovative techniques have been developed, in particular, in the domain of low-mass tracking devices. Detector construction spanned several years from 2009 to 2014. The collaboration started detector commissioning in 2014 and will collect data until the end of 2018. The beam line and detector components are described together with their early performance obtained from 2014 and 2015 data.NA62 is a fixed-target experiment at the CERN SPS dedicated to measurements of rare kaon decays. Such measurements, like the branching fraction of the $K^{+} \rightarrow \pi^{+} \nu \bar\nu$ decay, have the potential to bring significant insights into new physics processes when comparison is made with precise theoretical predictions. For this purpose, innovative techniques have been developed, in particular, in the domain of low-mass tracking devices. Detector construction spanned several years from 2009 to 2014. The collaboration started detector commissioning in 2014 and will collect data until the end of 2018. The beam line and detector components are described together with their early performance obtained from 2014 and 2015 data