Genes & Cells

The utilization of technology for the generation of induced pluripotent stem cells (iPSCs) and their subsequent differentiation is a promising approach for the study of disease pathogenesis and development of methods for treating optical neuropathies and retinopathy, which are the most common types of visual pathologies, in which retinal ganglion cells degenerate (consequently, optic nerve atrophy) or pigment epithelial cells and photoreceptors are affected, respectively. The prospect of patient-specific iPSCs has become a powerful alternative tool for discovering novel disease-causing mutations, studying genotype–phenotype relationships, screening therapeutic toxicity, and developing personalized cell therapy for optical neuropathies and retinopathies.

Numerous studies have demonstrated the possibility of creating different types of retinal cells from iPSCs, which provides a rapid development of the research area of human diseases for which no relevant animal models are available or access to primary human tissues and cells is limited.

This review presents various protocols for generating iPSCs from somatic cells and their subsequent differentiation, with an emphasis on the observed biological effects of the resulting cell cultures, including organoids, and discusses the prospects of using such models. The article may be useful to researchers studying the pathogenesis of various hereditary forms of blindness and developers of approaches for the treatment of these diseases who need a relevant cellular model.

BACKGROUND: Human genotype is a factor that determines the severity of COVID-19. Previously, a large-scale whole-genome association study of the COVID-19 Host Genetics Initiative (2021) investigated the association of genetic variants at multiple loci with COVID-19 severity. The genetic variants that have the greatest effect on COVID-19 severity are expected to have a low frequency in the population. Therefore, the study of rare variants may provide additional insights into the disease pathogenesis and thus help in the development of prevention and treatment options.

AIM: To search for genes enriched for rare genetic variants associated with COVID-19 severity in the Russian population by replication analysis.

METHODS: The clinical exome of a Russian cohort of patients was sequenced based on the St. Petersburg State Budgetary Institution “City Hospital No. 40” and St Petersburg University. The study used biomaterial from patients hospitalized at City Hospital No. 40 diagnosed with COVID-19 and healthy individuals (population control group). The severity of the course of COVID-19 was determined according to the results of lung computed tomography. The list of genes for subsequent replication was generated by a literature review. Burden test methods were used for the replication analysis of genes associated with COVID-19 severity.

RESULTS: In total, 701 clinical exomes were sequenced from 263 individuals with severe COVID-19 and 438 healthy individuals. In the literature review, 18 genes associated with severe COVID-19 were included in the replication analysis. The replication analysis did not identify any genes whose association with severe COVID-19 was confirmed in the study cohort.

CONCLUSION: The replication analysis did not identify any genes that showed a significant association between the functional variant enrichment and COVID-19 severity. However, the direction of the correlation was consistent with the findings of previous studies. Expanding the study cohort would increase the power of the tests and allow us to detect additional rare variants that influence the severity of COVID-19 progression.

BACKGROUND: When new, most often rare CFTR gene variants, are detected in patients with cystic fibrosis, intestinal organoids are used, which allows for the assessment of the residual functional activity of the CFTR channel and the effect of targeted drugs to determine the possibility of further pathogenetic therapy. Clinical trials of the effectiveness of the targeted drugs in patients with rare CFTR variants are expensive, and patient groups are extremely small. A forskolin-induced swelling assay on a patient’s intestinal organoids allows for a personalized approach when studying rare or even single CFTR variants.

AIM: To examine the effect of the CFTR potentiator ivacaftor and the combination of ivacaftor with the CFTR correctors lumacaftor, tezacaftor, and elexacaftor on the restoration of CFTR channel functions on a culture of intestinal organoids obtained from a patient with two rare CFTR variants c.264_268delATATT and c.3139+1G>C.

MATERIALS AND METHODS: To assess the activity of the CFTR channel, the forskolin-induced swelling assay on organoids and intestinal current measurements on rectal biopsy samples method were used. The clinical picture of a patient with the c.264_268delATATT/c.3139+1G>C genotype was described.

RESULTS: In vitro, the genetic variants c.264_268delATATT and c.3139+1G>C lead to a complete loss of the functional CFTR protein, whereas CFTR modulators do not positively affect and do not lead to the restoration of CFTR function, in contrast from F508del/F508del control. The disease severity in a child is consistent with the results of functional tests.

CONCLUSION: Both CFTR variants are classified as “severe” and cause a complete loss of chloride channel activity. No effective CFTR modulators were found for the c.264_268delATATT and c.3139+1G>C variants; thus, targeted therapy cannot be recommended to the patient.

BACKGROUND: The UBE2A protein belongs to the E2 family of ubiquitin-binding enzymes involved in the ubiquitination of substrate proteins. UBE2A mutations lead to congenital X-linked mental retardation syndrome-type Nascimento. How UBE2A participates in the central nervous system development is still unknown.

AIM: To establish a cell model based on induced pluripotent stem cells (iPSCs) to study the molecular and cellular functions of UBE2A in neurogenesis.

METHODS: Using genomic CRISPR-Cas9 editing and lentiviral transduction, a cell model based on iPSCs from two healthy donors was designed. This cell model includes isogenic iPSCs with knockout and inducible hyperexpression of UBE2A. In addition, iPSCs were obtained by reprogramming peripheral blood mononuclear cells of a patient diagnosed with X-linked mental retardation of Nascimento type, which has a deletion spanning the whole UBE2A locus.

RESULTS: The obtained iPSCs demonstrate an ESC-like morphology. They express pluripotent cell markers OCT4, SOX2, SSEA-4, and TRA-1-81 and have normal karyotypes. iPSCs with UBE2A knockout or hyperexpression had significantly increased nuclei size compared with the isogenic control.

CONCLUSION: The developed iPSC-based cell model can be used for fundamental studies of the functions of UBE2A in neurogenesis.