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Vol 11, No 2 (2016)

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Zakian S.M.
Genes & Cells. 2016;11(2):6-6
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Application of CRISPR/Cas9 system for developing and studying cellular models of inherited disease

Valetdinova K.R.


Currently, more than 4500 diseases classified as an inherited diseases. Most forms of human inherited diseases are caused by gene mutations. Given to the development of new methods genome editing, particularly with the use of CRISPR/Cas9 system, it is possible to artificially introduce precise mutations in the genome, as well as to correct gene mutations. It is thus possible to create a cellular model of human inherited diseases, as well as to carry out experiments for testing hypotheses and principles of gene and cell therapy. In this review, we consider studies that provide insight into the possibilities of CRISPR/Cas9 system for developing and studying cellular models of human inherited diseases.
Genes & Cells. 2016;11(2):10-20
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Site-specific genome editing for hematopoetic stem cells transplantation-based gene therapy approaches

Lepik K.V., Popova M.O., Shakirova A.I., Sergeev V.S., Potter A.Y., Barkhatov I.M., Fehse B., Afanasyev B.V.


Allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole universal curative approach for a number of hereditary diseases, such as severe combined immunodeficiency (SCID), severe non-SCID primary immunodeficiencies (non-SCID PID), hematologic diseases and part of lysosomal storage disorders. Unfortunately, to date, HSCT remains a high-risk procedure, especially in cases of poor performance status of the patient and lack of HLA-matched related donors. In those cases the correction of the patients autologous HSCs with gene therapy could be a promising alternative. Current paradigm of HSCT-based gene therapy approaches is based on the utilization of viral vectors, which may lead to the severe complications due to insertion mutagenesis. Throughout the last several years, new technologies of site-specific genome editing with endonucleases such as ZFNs, TALENs, and CRISPR/Cas9 were introduced. These enzymes may induce a DNA doublestranded break, homology-directed repair and insertion of functional copy of gene in precisely targeted locus. This review focuses on the advantages and disadvantages of the genome editing tools utilization that carries the great potential of changing the paradigm of gene therapy in the setting of HSCT.
Genes & Cells. 2016;11(2):21-31
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Methods of gene delivery and perspectives of their application in the gene therapy

Apartsin E.K., Knauer N.Y.


Gene therapy is believed to be among the most promising directions of the future medicine. Thus, the development of efficient and safe methods of the nucleic acid delivery to the target cells, tissues and organs ecomes of great current interest. This review summarizes recent data on the approaches for the gene delivery and discusses clinical aspects of the gene therapy.
Genes & Cells. 2016;11(2):32-41
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Preand posttranscriptional genetic information modification in muscular dystrophy treatment

Yakovlev I.A., Deev R.V., Solovyeva V.V., Rizvanov A.A., Isaev A.A.


Nowadays, a whole range of genetherapeutic methods is being used to restore a lost protein function due to mutation, a big number of preclinical and clinical studies of potential drugs that may allow to implement an etiotropic approach is being performed. 0ne of the most prevalent and socially significant groups of genetic pathologies is muscular dystrophy, including such diseases as Duchenne muscular dystrophy and dysfelinopathy. Despite a large number of studies in this field, there is no effective method of gene therapy for these diseases yet. This work is intended to review main genetherapeutic methods in myodystrophy treatment, especially pre- and posttranscriptional genetic (biosynthetic) information modification, and analyze most optimal of them.
Genes & Cells. 2016;11(2):42-52
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Editing of DNA epigenetic modifications

Grin I.R., Petrova D.V., Zharkov D.O.


Геномное редактирование в последние годы прочно вошло в арсенал методов молекулярной и клеточной биологии. Однако у высших эукариот огромную роль в формировании фенотипа клеток и организма в целом играет также состояние эпигенома - системы ковалентных модификаций ДНК и связанных с ней белков, регулирующих экспрессию генов. Методы редактирования эпигенома только начинают появляться, и в сочетании с технологиями геномного редактирования должны обеспечить более надежный контроль состояния изменяемых клеток. В обзоре освещены механизмы метилирования и активного деметилирования ДНК в клетках высших организмов и способы их применения для направленного изменения состояния эпигенома.
Genes & Cells. 2016;11(2):53-60
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The phenomenon of X chromosome inactivation and human diseases

Shevchenko A.I.


In early development, one of the two X chromosomes is randomly inactivated in each somatic cell of female embryos. As a result, women are mosaics that means about a half of their cells bear the active X chromosome inherited from the father, while the genes of the maternally inherited X chromosome are expressed in the other half. Disturbance in the inactivation process during embryogenesis leads to fetal death. Reactivation of the inactive X chromosome in female cells can cause a number of diseases, including cancer and autoimmune disorders. Changes in randomness of X-chromosome inactivation and skewed choice of one of the X-chromosomes for inactivation can influence clinical manifestations of about 400 diseases associated with mutations in X-linked genes. The phenomenon of X chromosome inactivation is also an important issue for successful application of human pluripotent stem cells in biomedical research and regenerative medicine.
Genes & Cells. 2016;11(2):61-69
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Neural differentiation of patient-specific induced pluripotent stem cells from patients with a hereditary form of spinal muscular atrophy

Grigor'eva E.V., Valetdinova K.R., Ustyantseva E.I., Shevchenko A.I., Medvedev S.P., Mazurok N.A., Maretina M.A., Kuranova M.L., Kiselev A.V., Baranov V.S., Zakian S.M.


Induced pluripotent stem cells (iPSCs) give the possibility for disease modeling, drug and toxicology screening and development of the new therapeutic approaches. Directed differentiation of iPSCs into specialized cell types represents a unique tool in order to study and model certain diseases, which affects specific type of cells, in vitro. One of the typical example of such diseases is spinal muscular atrophy, which is caused by mutations in the SMN1 gene (survival motor neuron 1 gene), leading to selective death of motor neurons. Patient-specific iPSCs were derived from the patient with a hereditary form of spinal muscular atrophy I type and expressed the markers of pluripotency (NANOG, TRA-1-60, SSEA4, OCT4, KLF4, MYC, REX1, and others). Spontaneous differentiation of the obtained cells resulted in the appearance of derivatives of the three germ layers: ecto-, meso- and endoderm. Neural differentiation showed the appearance of the early neural markers (PAX6, SOX2, NESTIN, TuJ1, PSA-NCAM), the late mature neural markers (MAP2, NF200, GFAP), and the mature motor neurons' markers (ISL1 and CHAT). Neurons derived from patient-specific iPSCs are perspective model for studying the features of the cells, which are altered in spinal muscular atrophy.
Genes & Cells. 2016;11(2):70-81
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Long noncoding RNA expression pattern in the X inactivation center of rodents

Elisaphenko E.A., Shevchenko A.I., Zakian S.M.


Transcriptome analysis of several cell lines with different differentiation degree has been carried out in three rodent species - voles, rats, and mice. As a result, expression profiles of X-inactivation center genes have been generated. In mice, new data on how expression of key genes of X-inactivation center, Xist and Tsix, changes during ontogenesis have been obtained. New types of transcripts have been revealed. In voles, Xist expression was observed in all 8 cell lines examined. Interestingly, main vole Xist transcript had 7 exons as those of rats and mice. In addition, Xist antisense transcript, Tsix, was expressed in 4 vole cell lines and was presented by both spliced and unspliced variants. Expression between the Enox (Jpx) and Xist genes has been revealed in three vole cell lines. Additional variants of Tsix transcripts were observed when studied expression profiles of several rat cell lines. The variants were not present in mice and voles. Compared to mice and voles, Tsix expression level in rats was several times higher than that of Xist.
Genes & Cells. 2016;11(2):82-86
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Strategies to edit paralogous genes with CRISPR/Cas9

Nemudryi A.A., Malankhanova T.B., Malakhova A.A., Medvedev S.P., Zakian S.M.


The purpose of this study is to develop the strategies of CRISPR/Cas9 application to improve fidelity and specificity of this platform. Here we use a model system, which includes target gene and a paralogue - potential aim for off-target double-strand break induction. The study was carried on using Brattleboro rats embryonic fibroblasts which are homozygous for a mutation in arginine-vasopressin gene (target). The potential off-target gene is oxytocin gene: its DNA sequence is almost identical to that of arginine-vasopressin gene. To prevent off-target effect we designed several strategies, which were further used on Brattleboro rats embryonic fibroblasts. Here we show, that these strategies allowed us to generate double-strand breaks in arginine-vasopressin gene without any off-target effects in oxytocin gene. The endonuclease restriction assay shows that we have modified arginine-vasopressin gene while using both CRISPR/Cas9 and single-stranded oligonucleotides as a donor for homologous recombination. At last, if we consider Brattleboro rats as a model of monogenic disease the strategies designed could be translated in human therapeutic genome editing studies.
Genes & Cells. 2016;11(2):87-94
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Plakophilin-2 gene knockout using CRISPR/Cas9 system

Khudiakov A.A., Kostina D.A., Kostareva A.A., Malashicheva A.B.


CRISPR/Cas9 technology opens up broad prospects for genome manipulations. Generation of gene knockout cell lines is one of the applications of this system. This approach allows to reveal previously unknown gene functions and to reproduce the effect of pathogenic mutations. The purpose of this study was to generate plakofilin-2 gene knockout system and assess its suitability for the study of the functional effect of this knockout.
Genes & Cells. 2016;11(2):95-99
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Delivery Cas9 into mitochondria

Orishchenko K.E., Sofronova J.K., Chupakhin E.G., Lunev E.A., Mazunin I.O.


Технологии направленного редактирования ядерного генома активно и успешно используются в лабораториях по всему миру. Нуклеазы на основе доменов типа «цинковые пальцы» (ZFN) и нуклеазы на основе эффекторных TAL-белков (TALENs) уже адаптированы для внесения двухцепочечных разрывов в митохондриальной ДНК (мтДНК). Появившаяся недавно более эффективная и универсальная технология CRISPR\Cas9 находится на начальном этапе её адаптации для мтДНК. Цель настоящей работы - модификация нуклеазы Cas9, одной из составляющих функциональных частей системы CRISPR\Cas9, для специфического импорта во внутримитохондриальное пространство. Адаптация второго компонента системы - направляющей РНК, позволяющая осуществлять ее специфическую доставку в митохондрии, даст возможность использовать данную систему для направленной деградации мтДНК содержащей мутации, а в перспективе позволит разработать подходы для её редактирования.
Genes & Cells. 2016;11(2):100-105
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Genome editing approach for generation of isogenic cell lines modelling Huntington's disease in vitro

Malakhova A.A., Sorokin M.A., Sorokina A.E., Malankhanova T.B., Mazurok N.A., Medvedev S.P., Zakian S.M.


Huntington's disease is an autosomal dominant neurodegenerative disorder caused by trinucleotide repeats expansion mutation in the first exon of HTT gene. Neurodegenerative disorders are difficult to study due to limited material availability and late onset of the manifestation. In vitro cell models provide a good opportunity to study molecular mechanisms of the pathology. New genome editing approaches allow us to generate isogenic cell lines that can be useful for drug screening and studying mechanisms of molecular and cellular events triggered by certain mutation on an equal genetic background. The aim of our work was establishing a method to introduce expanded CAG repeat tract into HTT gene by homology directed repair. We used a genome editing tool based on CRISPR/Cas9 system to facilitate the process. A donor construct bearing 215 CAG trinucleotide repeats flanked with long homology arms was generated. The method was approved on the HEK293Phoenix cell line. Mutant clones were obtained. It was shown that mutant HTT protein is expressed in the HEK293 mutant clones and affect their viability and proliferation rate.
Genes & Cells. 2016;11(2):106-113
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Genome editing on cell model of the genetic form of Parkinson's disease

Vetchinova A.S., Konovalova E.V., Volchkov P.Y., Abramycheva N.Y., Illarioshkin S.N.


New technological approaches for modeling of neurodegenerative disorders play growing role in modern neurobiology. One of such technologies is targeted genome editing with the use of artificial nuclease systems (CRISPR/ Cas9, etc.), that allow to correct genetic defects on the cellular level. Especially promising is the use of genome editing technology on specialized neurons and induced pluripotent stem cells obtained by cell reprogramming from patients with hereditary forms of neurodegenerative diseases. Using CRISPR/Cas9 system we performed editing of genome of induced pluripotent stem cells obtained from fibroblasts of a patient with autosomal recessive form of Parkinson's disease carrying compound heterozygous mutations in the PARK2 gene. Genome editing allowed to restore normal nucleotide sequence in both alleles of the PARK2 gene, correcting exonic (del202-203AG) and intronic (IVS1+1G/A) mutations. Possibility of genome editing and further obtaining of normal dopaminergic neurons from induced pluripotent stem cells is important both for studies of molecular mechanisms of neurodegenerative disorders and for successful development of cell therapy methods for Parkinson's disease.
Genes & Cells. 2016;11(2):114-118
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Optimization of teratoma formation assay

Kizilova E.A.


Teratoma formation assay is necessary to estimate in vivo pluripotency of stem cells especially stem cell lines of human origin. Nevertheless convenient, valid and universal “standards” to analyze stem cell derived tumors have not been developed yet. New protocol for monitoring teratoma growth, morphological and histological analyzes of tumor samples is proposed in this paper. This protocol is oriented on review of tumors morphology and histology per se. The list-describer includes 17 obligate and 12 facultative diagnostic sell types and 7 diagnostic cell complexes. The protocol takes into account complicity and heterogeneity of teratoma structure and allows detect different morphological features of malignization process inside stem cell derived tumors in situ. The protocol was successfully applied for teratoma formation test which has been performed for 52 stem cell lines of different species origin (mouse, rat, аmerican mink and human). 326 stem cell derived tumors were completely described, reviewed and analyzed.
Genes & Cells. 2016;11(2):119-128
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Genes & Cells. 2016;11(2):129-131
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