DNA nanotechnology has significantly advanced and might be used in biomedical applications, drug delivery, and cancer treatment during the past few decades. DNA nanomaterials are widely used in biomedical research involving biosensing, bioimaging, and drug delivery since they are remarkably addressable and biocompatible. Gradually, modified nucleic acids have begun to be employed to construct multifunctional DNA nanostructures with a variety of architectural designs. Aptamers are single-stranded nucleic acids (both DNAs and RNAs) capable of self-pairing to acquire secondary structure and of specifically binding with the target. Diagnosis and tumor therapy are prospective fields in which aptamers can be applied. Many DNA nanomaterials with three-dimensional structures have been studied as drug delivery systems for different anticancer medications or gene therapy agents. Different chemical alterations can be employed to construct a wide range of modified DNA nanostructures. Chemically altered DNA-based nanomaterials are useful for drug delivery because of their improved stability and inclusion of functional groups. In this work, the most common oligonucleotide nanomaterials were reviewed as modern drug delivery systems in tumor cells.

Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

New carriers for protein immobilization are objects of interest in various fields of biomedicine. Immobilization is a technique used to stabilize and provide physical support for biological micro- and macromolecules and whole cells. Special efforts have been made to develop new materials for protein immobilization that are non-toxic to both the body and the environment, inexpensive, readily available, and easy to modify. Currently, biodegradable and non-toxic polymers, including cellulose, are widely used for protein immobilization. Bacterial cellulose (BC) is a natural polymer with excellent biocompatibility, purity, high porosity, high water uptake capacity, non-immunogenicity, and ease of production and modification. BC is composed of glucose units and does not contain lignin or hemicellulose, which is an advantage allowing the avoidance of the chemical purification step before use. Recently, BC–protein composites have been developed as wound dressings, tissue engineering scaffolds, three-dimensional (3D) cell culture systems, drug delivery systems, and enzyme immobilization matrices. Proteins or peptides are often added to polymeric scaffolds to improve their biocompatibility and biological, physical–chemical, and mechanical properties. To broaden BC applications, various ex situ and in situ modifications of native BC are used to improve its properties for a specific application. In vivo studies showed that several BC–protein composites exhibited excellent biocompatibility, demonstrated prolonged treatment time, and increased the survival of animals. Today, there are several patents and commercial BC-based composites for wounds and vascular grafts. Therefore, further research on BC–protein composites has great prospects. This review focuses on the major advances in protein immobilization on BC for biomedical applications.

The maturation, development, and function of regulatory T cells (Tregs) are under the control of the crucial transcription factor Forkhead Box Protein 3 (FoxP3). Through alternative splicing, the human FoxP3 gene produces four different splice variants: a full-length variant (FL) and truncated variants with deletions of each of exons 2 (∆2 variant) or 7 (∆7 variant) or a deletion of both exons (∆2∆7 variant). Their involvement in the biology of Tregs as well as their association with autoimmune diseases remains to be clarified. The aim of this work was to induce a single FoxP3 splice variant in human Tregs by splice switching oligonucleotides and to monitor their phenotype and proliferative and suppressive activity. We demonstrated that Tregs from peripheral blood from patients with multiple sclerosis preferentially expressed truncated splice variants, while the FL variant was the major variant in healthy donors. Tregs with induced expression of truncated FoxP3 splice variants demonstrated lower suppressive activity than those expressing FL variants. Reduced suppression was associated with the decreased expression of Treg-associated suppressive surface molecules and the production of cytokines. The deletion of exons 2 and/or 7 also reduced the cell proliferation rate. The results of this study show an association between FoxP3 splice variants and Treg function and proliferation. The modulation of Treg suppressive activity by the induction of the FoxP3 FL variant can become a promising strategy for regenerative immunotherapy.

Forkhead box protein 3 (FoxP3) is a key transcription factor responsible for the development, maturation, and function of regulatory T cells (Tregs). The FoxP3 pre-mRNA is subject to alternative splicing, resulting in the translation of multiple splice variants. We have shown that Tregs from patients with amyotrophic lateral sclerosis (ALS) have reduced expression of full-length (FL) FoxP3, while other truncated splice variants are expressed predominantly. A correlation was observed between the reduced number of Tregs in the peripheral blood of ALS patients, reduced total FoxP3 mRNA, and reduced mRNA of its FL splice variant. Induction of FL FoxP3 was achieved using splice-switching oligonucleotides capable of base pairing with FoxP3 pre-mRNA and selectively modulating the inclusion of exons 2 and 7 in the mature mRNA. Selective expression of FL FoxP3 resulted in the induction of CD127^low, CD152, and Helios-positive cells, while the cell markers CD4 and CD25 were not altered. Such Tregs had an increased proliferative activity and a higher frequency of cell divisions per day. The increased suppressive activity of Tregs with the induced FL FoxP3 splice variant was associated with the increased synthesis of the pro-apoptotic granzymes A and B, and perforin, IL-10, and IL-35, which are responsible for contact-independent suppression, and with the increased ability to suppress telomerase in target cells. The upregulation of Treg suppressive and proliferative activity using splice-switching oligonucleotides to induce the predominant expression of the FoxP3 FL variant is a promising approach for regenerative cell therapy in Treg-associated diseases.

Background: Age plays a primary role in the development of age-related macular degeneration (AMD). Telomere length (TL) is one of the most relevant biomarkers of aging. In our study, we aimed to determine the association of TL with T lymphocytes, B lymphocytes, NK cells or monocytes with different forms of AMD. Methods: Our study included 62 patients with AMD: geographic atrophy (GA), neovascular AMD (NVAMD) with and without macular atrophy and 22 healthy controls. Each leukocyte subtype was isolated from peripheral blood by immunomagnetic separation, and the DNA was purified. The TL in the genomic DNA was determined using qPCR by amplifying the telomere region with specific oligonucleotide primers and normalizing to the control gene. Statistical analysis was performed using R version 4.5.1. Results: We observed a statistically significant increase in TL in the T cells between the control and NVAMD groups but not for the GA group. The B cells and monocytes showed a significant decrease in TL in all AMD groups. The TL in the NK cells did not decrease in any of the AMD groups. Conclusions: The TL in the monocytes had the strongest association with AMD. It reflects a person’s “telomeric status” and may become a diagnostic hallmark of these degenerative processes.