In silico analysis of a novel protein in CAR T- cell therapy for the treatment of hematologic cancer through molecular modelling, docking, and dynamics approach.

Cellular therapy has emerged as a key tool in the treatment of hematological malignancies. An advanced cell therapy known as chimeric antigen receptor T cell therapy (CAR T-cell therapy) has been approved by the United States Food and Drug Administration (FDA) as KYMRIAH by Novartis and YESCARTA by Gilead/Kite pharma in the year 2017. A chimeric receptor is composed of an extracellular antigen recognition site along with some co-stimulating and signalling domains. On the whole, it turns out to be one of the most potent receptors on T cells targeting a specific type of cancer cell based on its antigenic marker. CD19 CAR T-cell therapy is the first clinically approved therapy for lymphoma with remarkable results in complete remission of B cell lymphoblastic leukemia up to 90%. The high rate of effectiveness of the CAR T-cell therapy against B-ALL justifies the investigation and application of this therapy for fatal diseases like all types of hematological malignancies. The most critical aspect of chimeric receptor therapy is designing and building an artificial receptor that is specific to a given type of cancer. For this reason, the in silico technique is an appropriate model to investigate the integrity and effectiveness of the engineered chimeric receptor prior to commencing in vitro experiments followed by clinical trials. This computerized experimental study aids in predicting the molecular mechanism of chimeric protein and how it interacts with both ligands. We have anticipated various features of the chimeric protein in terms of qualitative analysis (structure, protein modelling, physiological properties) and functional analysis (antigenicity, allergenicity, its receptor-ligand binding ability, involving signalling pathways). Furthermore, the reliability and validation of the binding mode of the chimeric protein against receptors were performed through a complex molecular dynamics simulation for a 100 ns timeframe in an aqueous environment. The obtained simulation study showed that CD30 was a better approachable marker as compared to CD20 due to its better binding energy score and also binding conformations stability.

CRISPR-mediated knockdown of miR-214 modulates cell fate in response to anti-cancer drugs in HPV-negative and HPVpositive cervical cancer cells.

Cervical cancer is the fourth most common cause of mortality in women worldwide. In this study we investigated the effect of a tumour suppressor microRNA miR-214 in modulating the cell death against chemotherapeutic drugs like Doxorubicin, Cisplatin and Paclitaxel. CRISPR-facilitated knockdown and plasmid-based overexpression of miR-214 was performed in cervical cancer cell lines HeLa, C33A and CaSki. It was observed that knocking out miR-214 resulted in reduced apoptosis and cell migration upon drug treatments; while overexpression of miR-214 resulted in marginal increase in apoptosis and cell migration when treated with drugs. However, miR-214 had very little effect on production of reactive oxygen species. Our results also indicate that Doxorubicin was least effective and Paclitaxel most effective in inducing cell death. A combination of miR-214 overexpression and Paclitaxel treatment was found to be most effective in inducing cell death in cervical cancer cells. Analysis of cell cycle phases followed by apoptotic markers also showed that miR-214 overexpression along with Paclitaxel treatment caused an increase in PARP and decline of PI-3 kinase/Akt levels. Therefore, miR-214 levels determine the fate of the cancer cell during chemotherapeutic treatment.

Transcriptomic analyses of gene expression by CRISPR knockout of miR-214 in cervical cancer cells.

In this study, we investigate the effect of one such micro RNA, miR-214 which is frequently down-regulated in cervical cancer. In this study, we either CRISPR knocked out or overexpressed miR-214 in cervical cancer cells and analyzed the global mRNA expression by Next Generation Sequencing (NGS) It was observed that a total of 108 genes were upregulated and 178 downregulated between the samples, above and below the baseline respectively. Gene Ontology and KEGG pathway analysis reveal distinct biological processes and pathways. Analysis of gene regulatory networks also gave different network patterns in the two samples. We confirmed the RNA sequencing data for 10 genes; IFIF27, SMAD3, COX11, TP53INP1, ABL2, FGF8, TNFAIP3, NRG1, SP3 and MDM4 by Real-time PCR. This is the first report on the effect of miR-214 on global mRNA profile in cervical cancer cells. This study also reports new biomarkers for cervical cancer prognosis.

CAR T cell therapy: A new era for cancer treatment (Review).

Cancer has recently been identified as the leading cause of mortality worldwide. Several conventional treatments and cytotoxic immunotherapies have been developed and made available to the market. Considering the complex behavior of tumors and the involvement of numerous genetic and cellular factors involved in tumorigenesis and metastasis, there is a need to develop a promising immunotherapy that targets tumors at both the cellular and genetic levels. Chimeric antigen receptor (CAR) T cell therapy has emerged as a novel therapeutic T cell engineering practice, in which T cells derived from patient blood are engineered in vitro to express artificial receptors targeted to a specific tumor antigen. These directly identify the tumor antigen without the involvement of the major histocompatibility complex. The use of this therapy in the last few years has been successful, with a reduction in remission rates of up to 80% for hematologic cancer, particularly for acute lymphoblastic leukemia (ALL) and non­Hodgkin lymphomas, such as large B cell lymphoma. Recently, anti­CD19 CAR therapy, or UCART19, has been shown to be efficacious in treating relapsed/refractory hematologic cancer. Several other cell surface tumor antigens, such as CD20 and CD22, found in the majority of leukemias and lymphomas are considered potential targets by pharmaceutical companies and research organizations, and trials have been ongoing in this direction. Although this therapeutic regimen is currently confined to treating hematologic cancer, the increasing involvement of several auxiliary techniques, such as bispecific CAR, Tan­CAR, inhibitory­CAR, combined antigens, the clustered regularly interspaced short palindromic repeats gene­editing tool and nanoparticle delivery, may substantially improve its overall anticancer effects. CAR therapy has the potential to offer a rapid and safer treatment regime to treat non­solid and solid tumors. The present review presents an insight into the advantages and the advances of CAR immunotherapy and presents the emerging discrepancy of CAR therapy over usual forms of therapy, such as chemotherapy and radiotherapy.