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COMMON PART

Project Number17-75-20249

Project titleDevelopment of methods for modeling key aspects of the pathogenesis of human diseases in genetically modified animals

Project LeadDeykin Alexey

AffiliationInstitute of Gene Biology RAS,

Research area 05 - FUNDAMENTAL RESEARCH IN MEDICINE, 05-102 - Patanatomy, pathophysiology

KeywordsGenetically modified animal models, atherosclerosis, epileptic encephalopathy, Lesch-Naikhan syndrome, genomic editing, point mutations, mitochondrial genome

PROJECT CONTENT

Annotation
Genetically modified organisms are an indispensable tool for studying the functions of genes and non-coding sequences, the interaction of regulatory sequences in the genome and the expression of recombinant proteins, and also for modeling human diseases. Until recently, the production of genetically modified animals was very long and expensive, and therefore almost inaccessible to many groups of scientists, but everything changed with the advent of new systems for editing the genome. The first article on the application of the CRISPR / Cas9 system (clustered appropriately interspaced short palindromic repeats / CRISPR-associated nuclease 9) which allows in one step to inactivate multiple genes was published in 2013 [1]. The discovery of the CRISPR / Cas9 system has revolutionized production of genetically modified animals, shortening the duration of experiments from several years to several months. Since that time a new era has begun in which genetically modified organisms can be used not only by scientists in expensive experiments, but also by doctors in their practice when new genetic defects are discovered. The CRISPR / Cas9 system includes RNA containing regular clusters of short palindrome repeats (CRISPR), trans-activating RNA and the Cas9 protein (nuclease). This complex, which performs in nature the role of bacterial immunity against phage parasites [2], was adapted for editing DNA sequences both in vitro and in mammalian cells [3]. Genetically modified mice [4, 5], rats [5], monkeys [6], etc. have already been obtained by microinjection into the pronucleus of the zygotes of the RNA-guide complex with the CAS9 protein. We have shown that the insertion of both deletions and insertions occurs on mouse embryos with high efficiency and high accuracy [7]. In addition, we found that this system allows the introduction of single nucleotide substitutions in addition to extensive modifications (unpublished data). Currently, the sequencing of the genome and the identification of new mutations in the examination of patients is often of an analytical nature and only in rare cases helps to select the drug therapy more quickly. If there was a mouse model that duplicated the genetic defect of a specific patient, targeted therapy could be selected on it, varying compositions and doses of medications, minimizing the side effects of improperly selected combination and irreversible deterioration of the patient's condition. In addition, the availability of a mouse model will allow to test current drugs designed to correct a genetic defect, and not remote effects that develop in patients as a result of its presence. Such experiments are already conducted for other diseases, for example, Duchenne's myodystrophy and chronic ischemia of the lower limbs. In this project, there is the task to obtain genetically modified models of epileptic encephalopathy and Lesch-Nayhan syndrome, reproducing the genetic defect of specific patients, as well as two models of atherosclerosis, one of which due to a large number of errors during replication in mitochondria (including induced ones) will allow to select mice with mutations associated with atherosclerosis in humans, the other will allow testing the hypothesis about the participation of sialidases in the pathogenesis of atherosclerosis. An alternative strategy for obtaining mutations associated with atherosclerosis in patients will be development of technology for site-specific mutagenesis of mitochondrial genome due to the delivery to mitochondria of the Cas9-sgRNA complex and the template for homologous recombination at the site of gap. The introduction of point mutations into the nuclear and mitochondrial genome will be achieved by injecting into the zygotes of the mouse (into the pronucleus or cytoplasm) a CRISPR / Cas9-based system (with one or two target sgRNAs) and a matrix for repairing the gap (in the form of plasmid DNA, single-stranded DNA , DNA in combination with a signal of mitochondrial localization). The tasks will be carried out, as they are all technically feasible within the current level of science. The authors have experience in the creation of more than 120 lines of transgenic mice, including using CRISPR/Cas9 technology, as evidenced by publications in leading journals in the specialty, the successful implementation of projects of RNF and RFBR and State Contracts on relevant topics. An obstacle to obtaining three new models of deseases may be their lethality. On the one hand, it will still be the scientific results that can be documented and published, and, on the other hand, the use of modern approaches (site-specific insertion, targeted knockout of the analysis of the absence of mutations in the alternative sites induced, if necessary, double-lock "leakage" of the promoter, transgene expression) preclude failure of the project. The possibility of obtaining the results is based on the possession of a group of authors a technology of creation of genetically modified animals at the advanced level, the presence of own laboratory, constantly carrying out such work, full compliance with the material and technical basis of the current level of science and technology, the scale of the project objectives. At the same time, the team has the necessary expertise in the field of molecular and genetic mechanisms of the pathogenesis of model deseases the study of animal models and manifestations of the corresponding pathologies in humans. 1. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013; 153(4):910–918. PubMed PMID: 23643243 2. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816–821. 3. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819–823. 4. Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Scientific Reports. 2013; 3:3355. 5. Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M: Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol 2013, 31(8):681–683. 6. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J: Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014, 156(4):836–843. 7. Димитриева Т.В., Решетов Д.А., Жерновков В.Е., Влодавец Д.В., Зотова Е.Д., Ермолкевич Т.Г., Дейкин А.В. Модификация метода анализа результатов редактирования генома с помощью системы CRISPR/Cas9 на предимплантационных эмбрионах мыши. Вестник Российского государственного медицинского университета. 2016. № 3. С. 16-22. // 7. Dimitrieva TV, Reshetov DA, Zhernovkov VE, Vlodavets DV, Zotova ED, Ermolkevich TG, Deykin AV Modification of the method for analyzing the results of editing the genome using the CRISPR / Cas9 system on preimplantation mouse embryos. Bulletin of the Russian State Medical University. 2016. № 3. P. 16-22. 8. Jerry R Mendell and Louise R Rodino-Klapac: Duchenne muscular dystrophy: CRISPR/Cas9 treatment. Cell Research 2016, 26:513–514. doi:10.1038/cr.2016.28; 9. DE LOS ANGELES BEYTIA M, VRY J, KIRSCHNER J. Drug treatment of Duchenne muscular dystrophy: available evidence and perspectives. Acta Myologica. 2012;31(1):4-8. 10. Kadotani H, et al., Motor discoordination results from combined gene disruption of the NMDA receptor NR2A and NR2C subunits, but not from single disruption of the NR2A or NR2C subunit. J Neurosci. 1996 Dec 15;16(24):7859-67 11. Pierson, T. M., Yuan, H., Marsh, E. D., Fuentes-Fajardo, K., Adams, D. R., Markello, T., Gahl, W. A. (2014). GRIN2A mutation and early-onset epileptic encephalopathy: personalized therapy with memantine. Annals of Clinical and Translational Neurology, 1(3), 190–198. http://doi.org/10.1002/acn3.39 12. Бурлева Е.П., Бабушкина Ю.В. Применение препарата Неоваскулген для лечения больного с нейроишемической формой синдрома диабетической стопы Стр. 47-51 https://www.ncbi.nlm.nih.gov/pubmed/27336333 // 12. Burleva EP, Babushkina Yu.V. The use of Neovasculgen for the treatment of a patient with a neuroischemic form of diabetic foot syndrome Pp. 47-51 https://www.ncbi.nlm.nih.gov/pubmed/27336333

Expected results
As a result of the project, new genetic modified models of atherosclerosis, epileptic encephalopathy and Lesch-Nayhan syndrome will be obtained. Strains of mice will be obtained, the models are verified and transferred for preclinical testing to interested medical centers. The obtained results are the great importance for science and medicine, since not only new models of human diseases will be obtained, but a strategy of a personalized approach to modeling the disease and searching for therapy using an adequate genetic model of the pathological condition is worked out. The expected results correspond to the current level of biomedical research. It is supposed to use modern CRISPR / Cas9 technology of genomic editing and to obtain a model based on the genetic analysis of patients. These models are in demand by medical centers for treatment in which the relevant patients are located, but it is much more important to develop a strategy for treating difficult cases by modeling and screening potential medical strategies without injuring patients (including minors).

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