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Abstract Background Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models. Results pAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors. Conclusion Taken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.

Background: Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models. Results: pAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors. Conclusion: Taken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important. [ABSTRACT FROM AUTHOR]

Relevance. Online broadcasting and video recording of classes as a method of organizing e-learning and transferring knowledge using distance learning technologies today are of increasing interest to students and lecturers. However, most researchers distinguish the high labor intensity and the presence of additional skills on the part of the lecturer as a prerequisite for the implementation of a successful video lecture. At the same time, lecturers continue to conduct traditional classes in the “pre-pandemic” mode and the placement of additional material in the electronic information-educational environment (EIEE) for students on an individual schedule continues. Often, for remote participants in the educational process, an individual educational trajectory of learning is developed and the load on the lecturer increases, who is forced to use different methods and forms of education to ensure equal educational opportunities for all students. As an alternative for optimization, the authors propose the introduction of online broadcasting of classes as the most effective method and form of online learning.The purpose of the study is to develop a balanced model for the use of online broadcasting and video recording of classes in the organization of e-learning with the possibility of fixing the progress of the educational process and controlling knowledge.Materials and methods. Using a keyword search in the Russian Science Citation Index (RSCI) system, the authors found that researchers do not use the concept of “Online broadcasting of classes”. However, the analysis of publication activity in the RSCI system for 2017-2021 showed a positive trend in the use of the terms “Online lesson”, “Synchronous learning”, which are the closest in meaning to the concept of “Online broadcast of classes”. The publication proposes criteria for evaluating the effectiveness of the use of online broadcasting of classes in the organization of training at a university. As an example of the implementation of a solution for organizing online broadcasting, recording classes, the system “Information Resource Center” used in the Volga State University of Physical Culture, Sports and Tourism was described and compared with the proposed performance criteria.Results. As a result of the analysis, it was found that this type of systems (online broadcast systems) do not provide for the possibility of fixing the assimilation of the material and do not allow to fully fix the progress of the educational process and control knowledge when organizing e-learning.Conclusions. The authors propose the integration of online broadcast systems with the e-learning system as a working model for the use of online broadcast and video recording of classes in the organization of e-learning, which will satisfy all the criteria established in the study. The model designed in the course of the study is universal and can be used applying any methodological techniques, including the use of the “flipped class” model, when material for preliminary acquaintance is placed in advance of the lesson, and it is analyzed during the lesson itself.

Rotavirus has been associated with neonatal seizures and specific white matter magnetic resonance imaging (MRI) abnormalities. We describe monochorionic twins who not only tested positive for rotavirus with these white matter MRI abnormalities but who also showed an electroencephalogram (EEG) pattern characteristic of early infantile epileptic encephalopathy (EIEE), which has so far solely been described in epileptic encephalopathies with a poor prognosis. This report suggests that rotavirus infection must be added to the list of causes of EIEE EEG, and that the outcome then is likely more favorable. As MRI and EEG signs of rotavirus encephalopathy were present in one twin with only subtle neurologic symptoms, rotavirus may well cause insidious central nervous system complications more often. We suggest considering rotavirus infection in neonates presenting with seizures, and to add rotavirus infection to the differential diagnosis of EIEE. [ABSTRACT FROM AUTHOR]

Purpose: Epileptic encephalopathies (EE), are a group of age-related disorders characterized by intractable seizures and electroencephalogram (EEG) abnormalities that may result in cognitive and motor delay. Early infantile epileptic encephalopathies (EIEE) manifest in the first year of life. EIEE are highly heterogeneous genetically but a genetic etiology is only identified in half of the cases, typically in the form of de novo dominant mutations.Method: This is a descriptive retrospective study of a consecutive series of patients diagnosed with EIEE from the participating hospitals. A chart review was performed for all patients. The diagnosis of epileptic encephalopathy was confirmed by molecular investigations in commercial labs. In silico study was done for all novel mutations. A systematic search was done for all the types of EIEE and their correlated genes in the literature using the Online Mendelian Inheritance In Man and PubMed databases.Results: In this case series, we report 72 molecularly characterized EIEE from a highly consanguineous population, and review their clinical course. We identified 50 variants, 26 of which are novel, causing 26 different types of EIEE. Unlike outbred populations, autosomal recessive EIEE accounted for half the cases. The phenotypes ranged from self-limiting and drug-responsive to severe refractory seizures or even death.Conclusions: We reported the largest EIEE case series in the region with confirmed molecular testing and detailed clinical phenotyping. The number autosomal recessive predominance could be explained by the society's high consanguinity. We reviewed all the EIEE registered causative genes in the literature and proposed a functional classification. [ABSTRACT FROM AUTHOR]

Deletions in the 9q33-q34 region have been reported in patients with early onset epileptic encephalopathy, but a consistent phenotype has yet to emerge. We report on the diagnosis of a de novo 9q33-q34.12 microdeletion of 4 Mb in a 15-month-old girl presenting with severe psychomotor delay, facial dysmorphisms, thin corpus callosum and early myoclonic encephalopathy. This deletion encompasses 101 RefSeq genes, including the four autosomal dominant genes STXBP1, SPTAN1, ENG and TOR1A. We discuss genetic, clinical and epileptic features comparing our patient with those previously reported in the literature. [ABSTRACT FROM AUTHOR]

De novo STXBP1 mutations have been found in individuals with early infantile epileptic encephalopathy with suppression-burst pattern (EIEE). Our aim was to delineate the clinical spectrum of subjects with STXBP1 mutations, and to examine their biologic aspects. STXBP1 was analyzed in 29 and 54 cases of cryptogenic EIEE and West syndrome, respectively, as a second cohort. RNA splicing was analyzed in lymphoblastoid cells from a subject harboring a c.663 + 5G>A mutation. Expression of STXBP1 protein with missense mutations was examined in neuroblastoma2A cells. A total of seven novel STXBP1 mutations were found in nine EIEE cases, but not in West syndrome. The mutations include two frameshift mutations, three nonsense mutations, a splicing mutation, and a recurrent missense mutation in three unrelated cases. Including our previous data, 10 of 14 individuals (71%) with STXBP1 aberrations had the onset of spasms after 1 month, suggesting relatively later onset of epileptic spasms. Nonsense-mediated mRNA decay associated with abnormal splicing was demonstrated. Transient expression revealed that STXBP1 proteins with missense mutations resulted in degradation in neuroblastoma2A cells. Collectively, STXBP1 aberrations can account for about one-third individuals with EIEE (14 of 43). These genetic and biologic data clearly showed that haploinsufficiency of STXBP1 is the important cause for cryptogenic EIEE. [ABSTRACT FROM AUTHOR]

We, for the first time, used functional neuroimaging analyses for a girl with early myoclonic encephalopathy (EME). The interictal single photon emission computed tomography (SPECT) and [18F]-fluoro-d-deoxyglucose positron emission tomography (FDG-PET) at 1 month of age showed hypoperfusion and hypometabolism of bilateral basal ganglia, thalami, and the right parietooccipital cerebral cortices, showing that there is profound dysfunction of the basal ganglia and thalamus as well as cerebral cortex. On the other hand, subtraction ictal SPECT of tonic spasms clearly showed hyperperfusion of the bilateral basal ganglia, thalami, brainstem, and deep cortical layer of bilateral frontoparietal cortices. The present study suggests that functional deafferentation of the cortex from subcortical structures exists in EME, and that these imaging abnormalities may provide insight into the pathophysiology of suppression-burst pattern in EME. [ABSTRACT FROM AUTHOR]