Vol 11, No 2 (2016)

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.

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.

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.

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.

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.

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.

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.