Application of CRISPR–Cas9 technology in the treatment of chronic lymphocytic leukemia with mutant P53 gene

Abstract

Aim This study proposes the implementation of clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology for gene therapy targeting genetic mutations of P53 gene in human lymphocytes affected by chronic lymphocytic leukemia, (CLL), offering new opportunities for effective treatment of this heterogeneous disease. The objectives is related to the intended innovation, represent a continuation of the basic research and the project aim to establish, explore and implement this innovation potential of research results. Method CRISPR–Cas9 technology employs a specific enzyme guided by a designed guide RNA (gRNA) to a DNA target. The enzyme first introduces a cut at the target site and following this cleavage event, it can further disrupt the TP53 gene. The gRNA consists of CRISPR RNA (crRNA) and trans-activating CRISPR RNA, (tracrRNA), sequences, responsible for target recognition and Cas9 binding, respectively. Examination of the predicted secondary structure of the tracrRNA–crRNA duplex suggests that the features required for Cas9-catalyzed DNA cleavage at specific sites can be captured within a single chimeric RNA. Results Although the natural tracrRNA–crRNA mechanism operates efficiently, the use of a single RNA-guided Cas9 system is particularly attractive due to its potential for programmed DNA cleavage and genome editing. Importantly, Cas9 can bind and cleave a target sequence only if it is adjacent to a protospacer adjacent motif. Once the gRNA–Cas9 complex binds to the target DNA, Cas9 induces a double-strand break at the specified site, gene lesions, aiming to replace mutant TP53 genes in CLL cells through this technology. Conclusions CRISPR–Cas9 technology represents a powerful genetic engineering tool capable of inserting, deleting, or replacing DNA within an organism’s genome using these “molecular scissors.” 1. Introduction 1.1 Current state of research in the field & maturity of the project The present project aimed to implement the CRISPR/Cas9 (clustered regulated interspaced short palindromic repetitions) technology for gene therapy of genetic mutations of P53 gene in Chronic Lymphocytic Leukemia, (CLL) in human lymphocytes, offering new opportunities for an effective treatment in this heterogeneous disease. In this project, targeted sequencing using CRISPR/Cas9 approaches can used to systematically characterize the biological effects of monoallelic or biallelic P53 gene lesions, competing in CLL cells, and to replace the mutant P53 gene by this technology. Future perspectives: CRISPR in chronic lymphocytic leukemia The potential application of CRISPR technology in CLL follows models established in other hematologic disorders, such as β-thalassemia and sickle cell anemia severe hereditary hemoglobinopathies, where CRISPR–Cas9 has been successfully used for gene replacement and correction. These advances highlight CRISPR as a promising tool for targeted gene therapy of CLL. CRISPR systems have also been used during assay development and as plate controls in screening experiments. (1, 2, 3). 1.2 Own achievements in the field The diagnosed of CLL with mutant P53 gene can be analyzed by the Enzyme Linked Immune-Absorbent Assay (ELISA) technique, investigate the relationship of isoform p53 protein and survival patients before initiating any treatment. Sandwich ELISA, colorimetric quantitative method, can be used for direct detection of p53 isoform protein, product of Gene P-53: Specificity: human p53 protein (aa20-25); Format: Purified product: Monoclonal antibody clone: Isotype DO-1: IgG2a. The antibody is suitable for the techniques: ICC / IF and ELISA. Species reactivity is for human in conformity with the prospect from Manual, Catalog No. LS-F174. The research antibody PAb 240 antibodies bind specifically to denatured p53 protein as is the isoform p53 protein, in cytoplasmic product of mutant P53 gene. After analyzing the 85 LLC samples, in different stages of disease evolution, starting with stage zero (stay and watch) and up to stage IV, 20 patients were selected, eligible for this study, to be investigated for the detection of p53 protein isoforms responsible for resistance to oncological treatments of the disease with Rituximab, Cyclophosphamide, Doxorubicin hydrochloride , Vincristine sulfate (Oncovin), and Prednisone, (R-CHOP), after 2 cycles of relapses, representing a group of 16 men and 4 women aged 39-85 years. Male results: Protein concentration in p-53 / µg / dL: 20, 15, 18, 40, 10, 12, 14, 60, 30, 10, 13, 15, 5, 10, 15, 12. Women's results; Protein concentration p-53 / µg / dL: 140, 30, 13, 10. Normal values of normal cell lines on equipment: ELISA = 10 µg / dL, or 2.5- 5ng/mL. Very high pathological values in the 3 cases of p-53 were calculated in 2 men in the value of 60 µg / dL, respectively at 40 µg / dL, and in the case of females it calculated in the amount of 140 µg / dL, the frequency chronic lymphocytic leukemia with transformation into Diffuse Large Lymphoma, (DLL), DOI: https://doi.org/10.23958/ijirms/vol10-i05/2073 ], (4). Innovative potential and impact 1.3 Innovative potential The P53 chip can be tested in cooperation with partners at IARC (International Agency). In solid tumors, the P53 gene is mutated or deleted in approximately 50% of all tumors, but in leukemia, P53 gene aberrations are rare, ranging from 5-10% at the time of diagnosis (Peller & Rotter, 2003). Mutations in the P53 gene in CLL occur mainly in the DNA binding domain (exon 5-8), in the form of missense or nonsense mutations, deletions or insertions. Mutational analysis is performed on the entire cohort of CLL patients included in this genomic study. DNA can be isolated from MACS-sorted CD19+ B lymphocytes in the bone marrow. The Agilent Sure Select QXT Target Enrichment System for Illumina Multiplexed Sequencing (Agilent Technologies, Santa Clara, CA, USA) ca be used to produce libraries of exonic regions from CLL-related genes included in a previously validated, custom panel, (5). Mutations in the p53 gene of chromosome 17 (17p13.11) occur mostly in the DNA binding domain (exons 5-8), as missense or nonsense mutations, deletions or inserts. Like patients with 17p del, patients with mutant P53 genes show poor survival rates and resistance to chemotherapy. Among patients with chemotherapy-refractory disease, about 40-50% have lost one 17p allele or have a mutated p53 gene, leading to reduced overall survival rates. (Rossi et al., 2009). The 12 human isoform p53 protein have been identified (p53α, p53β, p53γ, ∆40p53α, ∆40p53β, ∆40p53γ, ∆133p53α, ∆133p53β, ∆133p53γ, ∆160p53α, ∆160p53β, and ∆160p53γ).[6]. Furthermore, p53 isoforms proteins are expressed in a tissue-dependent manner and P53α is never expressed alone. Missense mutations involved in the direct contact of P53 gene with DNA were adopted from the database of the International Agency for Research on Cancer, 1.4 Impact on economy, society CRISPR screening can identify the key genes predominantly influenced by single-gene disruption, the functional impact of many genes, their interactions, and the mutation profiles and genetic abnormalities of patients with significantly affect clinical outcomes. Findings highlight the essential role that CRISPR/Cas9-based synthetic lethal screens, this method, combined with clinical data, play as a powerful tool for gathering meaningful data on prognostic markers, novel therapeutic targets, mediators of drug response, and development approaches for novel drug combinations. 1.5 Sustainability Multiple hematological disorders are caused by known mutations in a single gene (Disease - GENE) affecting myeloid and/or lymphoid cells and may be suitable for CRISPR editing in HSCs. Scientists use nucleases that they have made to change the genome of induced pluripotent stem (iPS) cells. iPS cells are very useful for disease modelling and gene therapy because they can keep dividing and changing into different types of cells forever. 1.6 Intellectual Property (IP) situation Next-generation sequencing (NGS) analyzer and reagents as highly efficient genetic diagnostic test used in disease diagnosis and for the confirmatory diagnosis of CLL cases with the p53 protein isoform carried in the cell nucleus and cytoplasm of cells with the mutant p53 gene. Genomic DNA extracted from each sample is fragmented and used to construct a sequencing library with the Sure Select QXT Library Preparation Kit, following the manufacturer’s instructions. Reagents for the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) gene editing system which is a powerful tool for creating mutations and insertions in a cell's genomic DNA, (7). 2. Project plan 2.1 Scientific aims & challenges The present project aimed to implement the CRISPR/Cas9 (clustered regulated interspaced short palindromic repetitions) technology for gene therapy of genetic mutations in Chronic Lymphocytic Leukemia (CLL) in human lymphocytes, offering new opportunities for an effective treatment in this heterogeneous disease. In this project, targeted sequencing using CRISPR/Cas9 approaches was used to systematically characterize the biological effects of monoallelic or biallelic P53 gene The present project aimed to implement the CRISPR/Cas9 (clustered regulated interspaced short palindromic repetitions) technology for gene therapy of genetic mutations in Chronic Lymphocytic Leukemia, (CLL), in human lymphocytes, offering new opportunities for an effective treatment in this heterogeneous disease. In this project, targeted sequencing using CRISPR/Cas9 approaches can be used to systematically characterize the biological effects of monoallelic or biallelic P53 gene lesions, competing in CLL cells, and to replace the mutant P53 gene by this technology. 2.2 Methods & practical approach CRISPR–Cas9 technology employs a specific enzyme guided by a des