Generation of induced pluripotent stem cell line ICGi018-A from peripheral blood mononuclear cells of a patient with Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the HTT gene. HD patient-specific induced pluripotent stem cells (iPSCs) represent an excellent model for the disease study. We generated iPSC line from blood mononuclear cells of HD patient with 38 CAG repeats in the HTT exon 1 using integration free episomal plasmids expressing Yamanaka factors. The iPSC line retained the disease causing mutation and expressed pluripotency markers. It also displayed a normal karyotype and the ability to differentiate into derivatives of three germ layers.

Generation of an induced pluripotent stem cell line, ICGi014-A, by reprogramming peripheral blood mononuclear cells from a patient with homozygous D90A mutation in SOD1 causing Amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by death of motor neurons. To date, neither etiology nor pathogenesis of ALS are known, which leads to the absence of an effective treatment strategy. ALS patient-specific induced pluripotent stem cells (iPSCs) represent an excellent tool for the disease study. We obtained iPSCs line from peripheral blood mononuclear cells of the patient with homozygous Asp90Ala mutation in the SOD1 gene using non-integrating episomal vectors. The iPSCs line retained pathological genotype and expressed pluripotency markers. It also displayed a normal karyotype and the ability to differentiate into derivatives of three germ layers.

[Diagnostic algorithm for autosomal recessive ataxia]. / Algoritm diagnostiki autosomno-retsessivnykh ataksii.

AIM: To develop a complex algorithm for autosomal recessive ataxia (ARA) diagnosis applicable for Russian patients with degenerative ataxias. MATERIAL AND METHODS: 48 patients with of presumably degenerative ataxias were examined. Clinical evaluation was performed with the use of the SARA and ICARS scales (for ataxia) and MoCA (cognitive functions), and a set of laboratory tests was carried out, including electromyography, brain MRI, and DNA analysis of mutations responsible for Friedreich's disease and spinocerebellar ataxias (SCAs) types 1, 2, 3, 6 and 17. 28 patients underwent mutation screening using a multigenic MPS panel. RESULTS: 8 patients (16.7%) with non-hereditary causes of ataxia were identified: cerebellar alcoholic degeneration (n = 6) and multiple system atrophy of cerebellar type (n = 2); 3 patients (6.3%) with genetic ataxias were identified using routine DNA tests, such as with SCA type 1, 2 and 17, and 9 (18.8%) patients with Friedreich's disease. The MPS panel enabled molecular diagnosis of ARA in 8 patients (28.6%): ataxia-telangiectasia (n = 2), SANDO syndrome (n = 2), ataxia with oculomotor apraxia type 2 (n = 1), SCAR10 (n = 1), SCAR16 (n = 1), and atypical form of neuroaxonal dystrophy (n = 1). The diagnosis was not established in 20 patients. CONCLUSION: We have proposed an appropriate algorithm for degenerative ataxia diagnosis which is recommended to be used when examining patients with sporadic and autosomal recessive cases of the disorders with dyscoordination of movements.

A Platform for Studying Neurodegeneration Mechanisms Using Genetically Encoded Biosensors.

Patient-specific induced pluripotent stem cells (iPSCs) capable of differentiation into required cell type are a promising model for studying various pathological processes and development of new therapeutic approaches. However, no conventional strategies for using iPSCs in disease research have been established yet. Genetically encoded biosensors can be used for monitoring messenger molecules, metabolites, and enzyme activity in real time with the following conversion of the registered signals in quantitative data, thus allowing evaluation of the impact of certain molecules on pathology development. In this article, we describe the development of a universal cell-based platform for studying pathological processes associated with amyotrophic lateral sclerosis. For this purpose, we have created a series of plasmid constructs for monitoring endoplasmic reticulum stress, oxidative stress, apoptosis, and Ca2+-dependent hyperexcitability and generated transgenic iPSC line carrying mutation in the superoxide dismutase 1 gene (SOD1) and healthy control cell line. Both cell lines have specific transactivator sequence required for doxycycline-controlled transcriptional activation and can be used for a single-step biosensor insertion.

Genome Editing and the Problem of Tetraploidy in Cell Modeling of the Genetic Form of Parkinsonism.

The prevalent form of familial parkinsonism is caused by mutations in the LRRK2 gene encoding for the mitochondrial protein kinase. In the review, we discuss possible causes of appearance of tetraploid cells in neuronal precursors obtained from induced pluripotent stem cells from patients with the LRRK2-associated form of parkinsonism after genome editing procedure. As LRRK2 protein participates in cell proliferation and maintenance of the nuclear envelope, spindle fibers, and cytoskeleton, mutations in the LRRK2 gene can affect protein functions and lead, via various mechanisms, to the mitotic machinery disintegration and chromosomal aberration. These abnormalities can appear at different stages of fibroblast reprogramming; therefore, editing of the LRRK2 nucleotide sequence should be done during or before the reprogramming stage.

Cytogenetic Analysis of the Results of Genome Editing on the Cell Model of Parkinson's Disease.

We performed a cytogenetic analysis of the results of CRISPR/Cas9-correction of G2019S mutation in LRRK2 gene associated with Parkinson's disease. Genome editing was performed on induced pluripotent stem cells derived from fibroblasts of a patient carrying this mutation. A mosaic variant of tetraploidy 92 XXYY/46,XY (24-43% cells from various clones) was found in neuronal precursors differentiated from the induced pluripotent stem cells after gene editing procedure. Solitary cases of translocations and chromosome breaks were observed. These data confirm the importance of the development of new approaches ensuring genome stability in CRISPR/Cas9-edited cultures.

[Downregulation of human CCR5 receptor gene expression using artificial microRNAs].

Chemokine receptor CCR5 is essential for human immunodeficiency virus (HIV) entry into the sensitive cells. The CCR5 inactivation is believed to be one of the promising approaches in HIV therapy, including gene therapy. A powerful mechanism that enables to regulate gene expression is RNA interference which could be exploited to knockdown CCR5 gene. Here, three artificial microRNAs directed against the human CCR5 receptor gene were generated and their silencing activity in indicator cells developed on the basis of the HT1080 cell line was evaluated. Multiplexing of two or more artificial microRNAs in one transcript has been demonstrated to enhance the gene silencing. A 95% reduction of the CCR5 expression has been achieved using the most efficient microRNA combination.

Methods for identification of epigenetic elements in mammalian long multigenic genome sequences.

Epigenetic elements of the genome, i.e. elements that determine stably inherited changes in gene expression without changes in the genomic DNA sequence, are essential tools of genetic regulation in higher eukaryotes. The complete sequencing of the human and other genomes allowed studies to be started on positioning of these elements within long multigenic regions of the genome, which is a prerequisite for a comprehensive functional annotation of genomes. This mini-review considers some recent experimental approaches to the high-throughput identification and mapping of epigenetic elements of mammalian genomes, including the mapping of methylated CpG sites, open and closed chromatin regions, and DNase I hypersensitivity sites.

[Identification and mapping of cis-regulatory elements within long genomic sequences].

The publication of the human and other metazoan genome sequences opened up the possibility for mapping and analysis of genomic regulatory elements. Unfortunately, experimental data on genomic positions of such sequences as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. As most genomic regulatory elements (e.g., enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements in silico is often ambiguous. Therefore, the development of high-throughput experimental approaches for identification and mapping of genomic functional elements is highly desirable. In this review we discuss novel approaches to high-throughput experimental identification of mammalian genomes cis-regulatory elements which is a necessary step toward the complete genome annotation.

Tissue specificity of enhancer and promoter activities of a HERV-K(HML-2) LTR.

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.