Promoter Hypermethylation Promotes the Binding of Transcription Factor NFATc1, Triggering Oncogenic Gene Activation in Pancreatic Cancer

Studies have indicated that some genes involved in carcinogenesis are highly methylated in their promoter regions but nevertheless strongly transcribed. It has been proposed that transcription factors could bind specifically to methylated promoters and trigger transcription. We looked at this rather comprehensively for pancreatic ductal adenocarcinoma (PDAC) and studied some cases in more detail. Some 2% of regulated genes in PDAC exhibited higher transcription coupled to promoter hypermethylation in comparison to healthy tissue. Screening 661 transcription factors, several were found to bind specifically to methylated promoters, in particular molecules of the NFAT family. One of them—NFATc1—was substantially more strongly expressed in PDAC than control tissue and exhibited a strong oncogenic role. Functional studies combined with computational analyses allowed determining affected genes. A prominent one was gene ALDH1A3, which accelerates PDAC metastasis and correlates with a bad prognosis. Further studies confirmed the direct up-regulation of ALDH1A3 transcription by NFATc1 promoter binding in a methylation-dependent process, providing insights into the oncogenic role of transcription activation in PDAC that is promoted by DNA methylation.

PHENOMENON OF BIMODAL EXPRESSION OF WASP IN PATIENTS WITH WISKOTT–ALDRICH SYNDROME ASSESSED BY FLOW CYTOMETRY

Compensatory or revertant somatic mutations (RSM) is a well-known phenomenon in patients with PIs. RSM can lead to the restoration of functional protein expression in some cell populations and thus influence the severity of clinical manifestations in patients with PI. The WAS gene, a mutation that leads to the development of Wiskott–Aldrich syndrome (WAS), is associated with high levels of RSM. In our study, we aimed to describe in detail the RSM phenomenon in patients with WAS, and also to try to study its effect on the severity of the clinical phenotype. Materials and methods of research: a single-center, prospective, open, continuous, non-randomized, comparative study was conducted. The study included 39 male patients with WAS from 2 months and up to 18 years old with mutation in WAS gene, who underwent assessment of WASP expression using flow cytometry. In 2 out of 9 patients, use of magnetic selection made it possible to isolate a population of lymphocytes expressing WASP. Germinal and somatic mutations were identified using Sanger sequencing. To assess severity of clinical manifestations of WAS, the original detailed scoring classification was used. Results: 9 patients had bimodal expression of WASP, which equaled to 23% of all examined. The median age of these patients was 3 years [7 months. – 15 years]. In two patients a presence of RSM was confirmed in WASP-expressing populations. Linear assessment of partially restored WASP expression revealed different patterns of lymphocyte subpopulation involvement in all 9 patients. The effect of RSM on the severity of clinical manifestations of WAS in the studied group of patients was statistically insignificant (p=0,39). Conclusion: according to the data obtained, patients with WAS, demonstrating a bimodal pattern of WASP expression, represent a very heterogeneous group in terms of the severity of clinical manifestations, involvement of cell populations, and the nature of RSM. This work is a methodological prerequisite for further comprehensive study of RSM phenomenon in patients with various PIs.

Physiological (TCR-like) regulated lentiviral vectors for the generation of improved CAR-T cells

Background. Chimeric antigen receptor (CAR) T cells directed against CD19 have achieved impressive outcomes for the treatment of relapsed/refractory B lineage lymphoid neoplasms. However, CAR-T therapy still has important limitations due to severe side effects and the lack of efficiency in 40-50% of the patients. Most CARs-T products are generated using retroviral vectors with strong promoters. However, high CAR expression levels can lead to tonic signalling, premature exhaustion and over-stimulation of CAR-T cells, reducing efficacy and increasing side effects. TCR-like expression of the CAR through genome editing resulted in enhanced anti-tumour potency, reducing tonic signalling and improving CAR-T phenotype. In this manuscript, we searched for LVs that mimic the TCR expression pattern as a closer-to-clinic alternative for the generation of improved CAR-T cells. Methods. Different LVs containing viral and human promoters were analysed to select those that closely mimic a TCR-like kinetic profile upon T-cell activation. WAS gene proximal promoter-driven LVs (AW-LVs) were selected to express a second generation 4-1BB CD19 CAR (ARI-0001) into T cells to generate AWARI αCAR-T cells. TCR-like AWARI and EF1α-driven ARI CAR T cells were analysed for in vitro and in vivo killing efficiency using leukaemia and lymphoma cellular models. Tonic signalling, exhaustion markers and phenotype were determined by flow cytometry. Large-scale automated manufacturing of AWARI CAR-T cells was performed in a CliniMACs Prodigy bioreactor. Results. Our data showed that LVs expressing the transgene through the WAS gene proximal promoter mimic very closely the TCR (CD3) expression pattern kinetic upon TCR stimulation or antigen encounter. Compared to EF1α-driven ARI CAR-T cells, AWARI CAR-T cells exhibited a higher proportion of naive/stem cell memory T cells with less exhausted phenotype after efficient killing of CD19+ cells both in vitro and in vivo. AWARI CAR-T cells also showed lower tonic signalling and reduced secretion of pro-inflammatory cytokines and were efficiently manufactured in large-scale GMP-like conditions. Conclusions. WAS-gene-promoter driven LVs can be used to generate physiological 4-1BB-CAR-T cell products with lower tonic signalling, improved phenotype and a safer profile. We propose the use of TCR-like LVs as an alternative to strong-promoter driven LVs for the generation of CAR-T products.

Baboon Envelope Pseudotyped “Nanoblades” Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34+ Cells and Knock-in of AAV6-Encoded Donor DNA in CD34+ Cells

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into human blood cells can be challenging. Here, we have utilized “nanoblades,” a new technology that delivers a genomic cleaving agent into cells. These are modified murine leukemia virus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structural protein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 protein complexed with the guide RNAs. Highly efficient gene editing was obtained in cell lines, IPS and primary mouse and human cells. Here, we showed that nanoblades were remarkably efficient for entry into human T, B, and hematopoietic stem and progenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboon retroviral and VSV-G envelope glycoproteins. A brief incubation of human T and B cells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletion in the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34+ cells (HSPCs) treated with the same nanoblades allowed 30–40% exon 1 drop-out in the WAS gene locus. Importantly, no toxicity was detected upon nanoblade-mediated gene editing of these blood cells. Finally, we also treated HSPCs with nanoblades in combination with a donor-encoding rAAV6 vector resulting in up to 40% of stable expression cassette knock-in into the WAS gene locus. Summarizing, this new technology is simple to implement, shows high flexibility for different targets including primary immune cells of human and murine origin, is relatively inexpensive and therefore gives important prospects for basic and clinical translation in the area of gene therapy.

Gene flow in three broiler chicken populations in Abeokuta using jackknife procedure

Jackknife procedure (JP) is a less biased and fascinating method of obtaining gene slow among populations. The purpose of this study was to use JP to eliminate bias associated with indirect estimators of gene slow, with microsatellite markers, it has been possible to estimate gene flow (Nm) in natural populations. To quantify Nm, in chicken populations, we used five polymorphic microsatellite markers with 115 genomic deoxyribonucleic acid (DNA) obtained from (dihybrid (DH = 37), trihybrid (TH = 32) and Anak White (AW = 46) broiler chicken populations, respectively. Through polymerase chain reaction, we amplified DNA from the broiler chicken populations, subjected amplicons to electrophoresis, fragment sizes determined and analysed across populations considering selected markers through which standardized genetic variance among sub-populations in total sample depicted as (FST) was obtained per marker for chicken population pairs. Its average across markers/population pairs was used to infer Nm, in the chicken population pairs. We used JP which is a mathematical approach that utilizes mean FST, across markers to obtain Nm in the chicken population pairs. Gene flow based on JP in chicken population pairs designated as (Nm)JP were 5.4267 (DH vs. TH), 7.0127 (TH vs. AW) and 11.7405 (DH vs. AW) and among chicken populations, (Nm)JP was 7.1969. Considering these estimates, we concluded that there was gene flow among the three broiler chicken populations examined in Abeokuta, Nigeria.

Development of flow cytometry assay for Wiskott–Aldrich syndrome diagnosis by WASP protein evaluation

Wiskott–Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by microplatelet thrombocytopenia, eczema, frequent infections and an increased risk of autoimmune disorders and malignant neoplasms. Mutation detection in WAS gene is the gold standard for diagnosis of this disorder. This gene encodes a WASP protein, which works as regulator of cell cytoskeleton and is involved in the transmission of many intracellular signals. Nowadays there is no rapid and reliable method that allows to confirm WAS in a short period of time. Early detection of WAS in patients enables initiation of a donor search and preparation for the HSCT procedure. It also helps to avoid the development of severe and life-threatening conditions during waiting for genetic confirmation of the diagnosis by using pathogenetic therapy. Currently flow cytometry is one of the leading laboratory methods that permits to get the information about the expression of a protein in several hours. The study below describes rapid and reliable based on flow cytometry assay for WAS diagnosis. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. The study included 46 patients with suspected WAS from 2 months to 17 years old. Patients were examined from January 2018 to January 2020. WAS gene defect was confirmed in 35 patients. It was calculated that normal threshold value for WASP expression is 7.07 with sensitivity and specificity 100% and 93.1% respectively. Besides negative correlation between WASP expression index and WAS clinical severity was shown (r = –0.63). This flow cytometry assay can be used for chimerism detection in WAS patients after HSCT. The flow cytometry assay for WASP protein evaluation is rapid, highly sensitive and highly specific. It allows to speed up diagnosis of this disorder.

Interpreter Sam

As a new hire, Sam was assigned to an exploration team tasked with evaluating the resource potential of an undrilled frontier basin in preparation for nomination of acreage for a lease sale. His boss was Gene, a geophysicist who had worked his way up the ladder to a team leader position and enjoyed a reputation as a tough but fair supervisor with a solid technical foundation. His team consisted of three geophysicists/seismic interpreters, two geologists, and one reservoir engineer, this composition reflecting the size and complexity of the basin and the importance of seismic interpretation to the project. As the junior interpreter, Sam worked with the more experienced interpreters in an apprentice-master relationship, and remembers his experience on this project as some of the most formative of his career.

The phenomenon of reverse mutation in a patient with Wiskott–Aldrich syndrome

Primary immunodeficiencies (PIDS) are genetically caused heterogeneous diseases of the immune system. One of the genetic phenomenon affecting the phenotypic diversity of PIDS is a reverse somatic mosaicism (RM) observed in different groups of PIDS. The majority of RM cases are described in patients with Wiskott–Aldrich syndrome (WAS). Despite the fact that PM does not always lead to a mild form of the disease, the presence of this phenomenon can cause the delay of diagnosis and start of the appropriate treatment. This article presents the case of a patient with Wiskott–Aldrich syndrome with a reverse mutation in the WAS gene. Parents gave their consent to use information about the child in the article.