Gene Therapy Approaches for the Treatment of Hemophilia B.

In contrast to the standard enzyme-replacement therapy, administered from once per 7-14 days to 2-3 times a week in patients with severe hemophilia B, as a result of a single injection, gene therapy can restore F9 gene expression and maintain it for a prolonged time. In clinical research, the approach of delivering a functional copy of a gene using adeno-associated viral (AAV) vectors is widely used. The scientific community is actively researching possible modifications to improve delivery efficiency and expression. In preclinical studies, the possibility of genome editing using CRISPR/Cas9 technology for the treatment of hemophilia B is also being actively studied.

Cap-Independent Circular mRNA Translation Efficiency.

Recently, the mRNA platform has become the method of choice in vaccine development to find new ways to fight infectious diseases. However, this approach has shortcomings, namely that mRNA vaccines require special storage conditions, which makes them less accessible. This instability is due to the fact that the five-prime and three-prime ends of the mRNA are a substrate for the ubiquitous exoribonucleases. To address the problem, circular mRNAs have been proposed for transgene delivery as they lack these ends. Notably, circular RNAs do not have a capped five-prime end, which makes it impossible to initiate translation canonically. In this review, we summarize the current knowledge on cap-independent translation initiation methods and discuss which approaches might be most effective in developing vaccines and other biotechnological products based on circular mRNAs.

Robust AAV Genotyping Based on Genetic Distances in Rep Gene That Are Maintained by Ubiquitous Recombination.

Adeno-associated viruses (AAVs) are a convenient tool for gene therapy delivery. According to the current classification, they are divided into the species AAV A and AAV B within the genus Dependoparvovirus. Historically AAVs were also subdivided on the intraspecies level into 13 serotypes, which differ in tissue tropism and targeted gene delivery capacity. Serotype, however, is not a universal taxonomic category, and their assignment is not always robust. Cross-reactivity has been shown, indicating that classification could not rely on the results of serological tests alone. Moreover, since the isolation of AAV4, all subsequent AAVs were subdivided into serotypes based primarily on genetic differences and phylogenetic reconstructions. An increased interest in the use of AAV as a gene delivery tool justifies the need to improve the existing classification. Here, we suggest genotype-based AAV classification below the species level based on the rep gene. A robust threshold was established as 10% nt differences within the 1248 nt genome fragment, with 4 distinct AAV genotypes identified. This distinct sub-species structure is maintained by ubiquitous recombination within, but not between, rep genes of the suggested genotypes.

Universal Flu mRNA Vaccine: Promises, Prospects, and Problems.

The seasonal flu vaccine is, essentially, the only known way to prevent influenza epidemics. However, this approach has limited efficacy due to the high diversity of influenza viruses. Several techniques could potentially overcome this obstacle. A recent first-in-human study of a chimeric hemagglutinin-based universal influenza virus vaccine demonstrated promising results. The coronavirus pandemic triggered the development of fundamentally new vaccine platforms that have demonstrated their effectiveness in humans. Currently, there are around a dozen messenger RNA and self-amplifying RNA flu vaccines in clinical or preclinical trials. However, the applicability of novel approaches for a universal influenza vaccine creation remains unclear. The current review aims to cover the current state of this problem and to suggest future directions for RNA-based flu vaccine development.

A critical role for regulatory T cell-mediated control of inflammation in the absence of commensal microbiota.

Suppression mediated by regulatory T cells (T reg cells) represents a unique, cell-extrinsic mechanism of in-trans negative regulation that restrains multiple types of immune cells. The loss of T reg cells leads to fatal, highly aggressive, and widespread immune-mediated lesions. This severe autoimmunity may be driven by commensal microbiota, the largest source of non-self ligands activating the innate and adaptive immune systems. Alternatively, T reg cells may primarily restrain T cells with a diverse self-major histocompatibility complex (MHC)-restricted T cell receptor repertoire independently of commensal microbiota. In this study, we demonstrate that in germ-free (GF) mice, ablation of the otherwise fully functional T reg cells resulted in a systemic autoimmune lympho- and myeloproliferative syndrome and tissue inflammation comparable with those in T reg cell-ablated conventional mice. Importantly, there were two exceptions: in GF mice deprived of T reg cells, the inflammation in the small intestine was delayed, whereas exocrine pancreatitis was markedly accelerated compared with T reg cell-ablated conventional mice. These findings suggest that the main function of T reg cells is restraint of self-MHC-restricted T cell responsiveness, which, regardless of the presence of commensal microbiota, poses a threat of autoimmunity.

Efficient differentiation of hESCs into endothelial cells in vitro is secured by epigenetic changes.

Human embryonic stem cells (hESCs) are to be considered as a valuable source for regenerative medicine because of their capacity to differentiate into all cell types. We have developed an efficient culture system to differentiate hECSs into endothelial cells without the formation of embryoid bodies Establishing appropriate culture conditions with a cocktail of growth factors allowed us to differentiate hESCs directly to endothelial primary culture with about 50% efficiency. CD31 immunomagnetic cell sorting was used to purify derived endothelium from the primary culture of hESCs. Isolated endothelial cells expressed immunological markers (vWF, CD105), specific genes (VE-cadherin, KDR, GATA-2, GATA-3, eNOS), and formed cord-like structures on collagen matrix and in Matrigel assay. During differentiation to endothelial lineage promoter regions of the genes involved in specific cell fate determination and homeostasis (GATA-2,-3, and eNOS) underwent intensive hypomethylation which correlated with the gene expression. Overall our data demonstrate that direct differentiation of hESCs leads to endothelial cells that acquire epigenetic patterning similar to the functional endothelial cells of the organism.

Diverse epigenetic profile of novel human embryonic stem cell lines.

Human embryonic stem cells (hESCs) are a promising model for studying mechanisms of regulation of early development and differentiation. OCT4, NANOG, OCT4-related genes and some others were recently described to be important in pluripotency maintenance. Lesser is known about molecular mechanisms involved in their regulation. Apart from genetic regulation of gene expression epigenetic events, particularly methylation, play an important role in early development. Using RT-PCR we studied the expression of pluripotency-related genes OCT4, NANOG, DPPA3 and DPPA5 during hESCs differentiation to embryoid bodies. Analysis of methylation profiles of promoter or putative regulatory regions of the indicated genes demonstrated that expression of the pluripotency-maintaining genes correlated with their methylation status, whereas methylation of DPPA3 and DPPA5 varied between cell lines. We propose that DNA methylation underlies the developmental stage-specific mechanisms of pluripotency-related genes expression and reactivation and may have an impact on differentiation potential of hESC lines.