Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases.

The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.

The Development, Optimization and Future of Prime Editing.

Prime editing is a rapidly developing method of CRISPR/Cas-based genome editing. The increasing number of novel PE applications and improved versions demands constant analysis and evaluation. The present review covers the mechanism of prime editing, the optimization of the method and the possible next step in the evolution of CRISPR/Cas9-associated genome editing. The basic components of a prime editing system are a prime editor fusion protein, consisting of nickase and reverse transcriptase, and prime editing guide RNA, consisting of a protospacer, scaffold, primer binding site and reverse transcription template. Some prime editing systems include other parts, such as additional RNA molecules. All of these components were optimized to achieve better efficiency for different target organisms and/or compactization for viral delivery. Insights into prime editing mechanisms allowed us to increase the efficiency by recruiting mismatch repair inhibitors. However, the next step in prime editing evolution requires the incorporation of new mechanisms. Prime editors combined with integrases allow us to combine the precision of prime editing with the target insertion of large, several-kilobase-long DNA fragments.

Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7.

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.

Methods for CRISPR-Cas as Ribonucleoprotein Complex Delivery In Vivo.

The efficient delivery of CRISPR-Cas components is still a key and unsolved problem. CRISPR-Cas delivery in the form of a Cas protein+sgRNA (ribonucleoprotein complex, RNP complex), has proven to be extremely effective, since it allows to increase on-target activity, while reducing nonspecific activity. The key point for in vivo genome editing is the direct delivery of artificial nucleases and donor DNA molecules into the somatic cells of an adult organism. At the same time, control of the dose of artificial nucleases is impossible, which affects the efficiency of genome editing in the affected cells. Poor delivery efficiency and low editing efficacy reduce the overall potency of the in vivo genome editing process. Here we review how this problem is currently being solved in scientific works and what types of in vivo delivery methods of Cas9/sgRNA RNPs have been developed.

Studies on glycogen storage disease type 1a animal models: a brief perspective.

Glycogen storage disease type 1 (GSD1) is a rare hereditary monogenic disease characterized by the disturbed glucose metabolism. The most widespread variant of GSD1 is GSD1a, which is a deficiency of glucose-6-phosphatase-ɑ. Glucose-6-phosphatase-ɑ is expressed only in liver, kidney, and intestine, and these organs are primarily affected by its deficiency, and long-term complications of GSD1a include hepatic tumors and chronic liver disease. This article is a brief overview of existing animal models for GSD1a, from the first mouse model of 1996 to modern CRISPR/Cas9-generated ones. First whole-body murine models demonstrated exact metabolic symptoms of GSD1a, but the animals did not survive weaning. The protocol for glucose treatment allowed prolonged survival of affected animals, but long-term complications, such as hepatic tumorigenesis, could not be investigated. Next, organ-specific knockout models were developed, and most of the metabolic research was performed on liver glucose-6-phosphate-deficient mice. Naturally occuring mutation was also discovered in dogs. All these models are widely used to study GSD1a from metabolic and physiological standpoints and to develop possible treatments involving gene therapy. Research performed using these models helped elucidate the role of glycogen and lipid accumulation, hypoxia, mitochondrial dysfunction, and autophagy impairment in long-term complications of GSD1a, including hepatic tumorigenesis. Recently, gene replacement therapy and genome editing were tested on described models, and some of the developed approaches have reached clinical trials.

Rational Design of ssODN to Correct Mutations by Gene Editing.

Gene editing allows to make a variety of targeted changes in genome, which can potentially be used to treat hereditary human diseases. Despite numerous studies in this area, effectiveness of gene editing methods for correcting mutations is still low, so these methods are not allowed in routine practice. It has been shown that rational design of genome editing components can significantly increase efficiency of mutation correction. In this work, we propose design of single-stranded oligodeoxyribonucleotides (ssODNs) to increase efficiency of gene editing. Using a model system to repair knocked out EGFP that is integrated into the genome of HEK293T cell culture, we have shown that only a small part of ssODN (about 20 nucleotides: from the 15th nucleotide at 3'-end to the 4th nucleotide at 5'-end), a donor molecule for repairing double-stranded DNA breaks, is integrated into the site of the break. Based on the obtained data, it is possible to rationally approach the design of ssODNs to correct mutations using CRISPR-Cas9 method for the development of gene therapy for hereditary human diseases.

Perspectives on Genetic Medicine for Cystic Fibrosis.

Like any inherited protein deficiency disease, cystic fibrosis (CF) is a good candidate for gene replacement therapy. Despite the tremendous efforts of scientists worldwide invested in developing this approach, it did not lead to the expected results for various reasons discussed in this review. At the same time, the emergence of new methods of genome editing, as well as their latest modifications, makes it possible to bypass some of the problems of "classical" CF gene therapy. The review examines potential therapeutic agents for CF gene therapy, methods and routes of delivery, as well as discusses the problem of target cells for defect correction. Based on the results of these studies, editing genetic defects in the basal cells of the lungs and their counterparts in other organs will make it possible to create a drug for treating CF with a single administration.

Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle.

Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be extremely important and promising. Numerous attempts to improve the efficiency of homology-directed repair-mediated correction of mutations in mammalian cells have focused on influencing the cell cycle. Homology-directed repair is known to occur only in the late S and G2 phases of the cell cycle, so researchers are looking for safe ways to enrich the cell culture with cells in these phases of the cell cycle. This review surveys the main approaches to influencing the cell cycle in genome editing experiments (predominantly using Cas9), for example, the use of cell cycle synchronizers, mitogens, substances that affect cyclin-dependent kinases, hypothermia, inhibition of p53, etc. Despite the fact that all these approaches have a reversible effect on the cell cycle, it is necessary to use them with caution, since cells during the arrest of the cell cycle can accumulate mutations, which can potentially lead to their malignant transformation.

Off-target effects of base editors: what we know and how we can reduce it.

The recently discovered CRISPR-Cas9 modification, base editors (BEs), is considered as one of the most promising tools for correcting disease-causing mutations in humans, since it allows point substitutions to be edited without generating double-stranded DNA breaks, and, therefore, with a significant decrease in non-specific activity. Until recently, this method was considered the safest, but at the same time, it is quite effective. However, recent studies of non-specific activity of BEs revealed that some of them lead to the formation of a huge number of off-targets in both DNA and RNA, occurring due to the nature of the Cas9-fused proteins used. In this review article, we have considered and combined data from numerous studies about the most commonly used and more described in detail APOBEC-based BEs and Target-AID version of CBE, as well as ABE7 and ABE8 with their basic modifications into TadA to improve BEs' specificity. In our opinion, modern advances in molecular genetics make it possible to dramatically reduce the off-target activity of base editors due to introducing mutations into the domains of deaminases or inhibition of Cas9 by anti-CRISPR proteins, which returns BEs to the leading position in genome editing technologies.

P.F508del editing in cells from cystic fibrosis patients.

Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out.

Exome, transcriptome and miRNA analysis don't reveal any molecular markers of TKI efficacy in primary CML patients.

BACKGROUND: Approximately 5-20% of chronic myeloid leukemia (CML) patients demonstrate primary resistance or intolerance to imatinib. None of the existing predictive scores gives a good prognosis of TKI efficacy. Gene polymorphisms, expression and microRNAs are known to be involved in the pathogenesis of TKI resistance in CML. The aim of our study is to find new molecular markers of TKI therapy efficacy in CML patients. METHODS: Newly diagnosed patients with Ph+ CML in chronic phase were included in this study. Optimal and non-optimal responses to TKI were estimated according to ELN 2013 recommendation. We performed genotyping of selected polymorphisms in 62 blood samples of CML patients, expression profiling of 33 RNA samples extracted from blood and miRNA profiling of 800 miRNA in 12 blood samples of CML patients. RESULTS: The frequencies of genotypes at the studied loci did not differ between groups of patients with an optimal and non-optimal response to TKI therapy. Analysis of the expression of 34,681 genes revealed 26 differently expressed genes (p < 0.05) in groups of patients with different TKI responses, but differences were very small and were not confirmed by qPCR. Finally, we did not find difference in miRNA expression between the groups. CONCLUSIONS: Using modern high-throughput methods such as whole-exome sequencing, transcriptome and miRNA analysis, we could not find reliable molecular markers for early prediction of TKI efficiency in Ph+ CML patients.

Ways of improving precise knock-in by genome-editing technologies.

Despite the recent discover of genome-editing methods, today we can say these approaches have firmly entered our life. Two approaches-knocking out malfunctioning gene allele or correcting the mutation with precise knock-in-can be used in hereditary monogenic diseases treatment. The latter approach is relatively ineffective. Modern data about the ways of repair of double-strand DNA breaks formed by nucleases are presented in this review. The main part of the review is devoted to the ways of increasing precise and effective knock-in: inhibition of non-homologous end joining and stimulation of homology-directed repair key factors, use of small molecules with unknown mechanism of action, cell-cycle synchronization and cell-cycle-dependent activity of Cas9, donor molecule design, selection, alternative methods for insertion and other approaches.

Copy number variation analysis in cytochromes and glutathione S-transferases may predict efficacy of tyrosine kinase inhibitors in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of BCR/ABL fusion gene in leukemic cells, which promotes uncontrolled cell proliferation. Up to 20% of CML patients show primary resistance or non-optimal response to tyrosine kinase inhibitor (TKI) therapy. We investigated the association between copy number variation (CNV) in glutathione S-transferases (GST) and cytochromes (CYP) and the response rate to TKI. We enrolled 47 patients with CML: 31 with an optimal response and 16 with failure at 6 months in accordance with European LeukemiaNet 2013 recommendations. CNV detection was performed using SALSA MLPA P128-C1 Cytochrome P450 probe mix. Patients with optimal response and with failure of TKI therapy showed different frequencies of wild type and mutated CYPs and GST (p<0.0013). Validation in the group of 15 patients proved high prognostic value (p = 0.02): positive and negative predictive value 83% and 78%; sensitivity and specificity 71% and 88%. Wild type genotypes of CYP and GST associate with a worse response to TKI treatment in CML patients. This test can be recommended for further clinical trials.

Frequent variations in cancer-related genes may play prognostic role in treatment of patients with chronic myeloid leukemia.

BACKGROUND: Genome variability of host genome and cancer cells play critical role in diversity of response to existing therapies and overall success in treating oncological diseases. In chronic myeloid leukemia targeted therapy with tyrosine kinase inhibitors demonstrates high efficacy in most of the patients. However about 15 % of patients demonstrate primary resistance to standard therapy. Whole exome sequencing is a good tool for unbiased search of genetic variations important for prognosis of survival and therapy efficacy in many cancers. We apply this approach to CML patients with optimal response and failure of tyrosine kinase therapy. RESULTS: We analyzed exome variations between optimal responders and failures and found 7 variants in cancer-related genes with different genotypes in two groups of patients. Five of them were found in optimal responders: rs11579366, rs1990236, rs176037, rs10653661, rs3803264 and two in failures: rs3099950, rs9471966. These variants were found in genes associated with cancers (ANKRD35, DNAH9, MAGEC1, TOX3) or participating in cancer-related signaling pathways (THSD1, MORN2, PTCRA). CONCLUSION: We found gene variants which may become early predictors of the therapy outcome and allow development of new early prognostic tests for estimation of therapy efficacy in CML patients. Normal genetic variation may influence therapy efficacy during targeted treatment of cancers.

Whole-exome sequencing reveals potential molecular predictors of relapse after discontinuation of the targeted therapy in chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML) is a myeloproliferative disease well treated by tyrosine kinase inhibitors (TKIs). The aim was to identify genes with a predictive value for relapse-free survival after TKI cessation in CML patients. We performed whole-exome sequencing of DNA from six CML patients in long-lasting deep molecular remission. Patients were divided into two groups with relapse (n = 3) and without relapse (n = 3) after TKI discontinuation. We found variants in genes CYP1B1, ALPK2, and IRF1 in group of patients with relapse and one variant in gene PARP9 in group of patients without relapse. We verified prognostic value of the found markers in a small group of patients with TKI discontinuation and demonstrated their high sensitivity (77%), specificity (86%), positive (85%), and negative (79%) predictive values. Thus we revealed genetic variants, which are potential markers of outcome prediction in CML patients after TKI discontinuation.