[Effective Viral Delivery of Genetic Constructs to Neuronal Culture for Modeling and Gene Therapy of GNAO1 Encephalopathy].

GNAO1 encephalopathy is an orphan genetic disease associated with early infantile epilepsy, impaired motor control, and severe developmental delay. The disorder is caused by mutations in the GNAO1 gene, leading to dysfunction of the encoded protein Gao1. There is no cure for this disease, and symptomatic therapy is ineffective. Phenotypic heterogeneity highlights the need for a personalized approach for treating patients with a specific clinical variant of GNAO1 and requires the study of the disease mechanism in animal and cell models. Towards this aim, we developed an approach for modeling GNAO1 encephalopathy and testing gene therapy drugs in primary neurons derived from healthy mice. We optimized the delivery of transgenes to Gαo1-expressing neurons using recombinant adeno-associated viruses (rAAV). We assessed the tropism of five neurotropic AAV serotypes (1, 2, 6, 9, DJ) for Gαo1-positive neurons from the whole mouse brain. The DJ serotype showed the highest potential as a reporter delivery vehicle, infecting up to 66% of Gαo1-expressing cells without overt cytotoxicity. We demonstrated that AAV-DJ also provides efficient delivery and expression of genetic constructs encoding normal and mutant Gαo1, as well as short hairpin RNA (shRNA) to suppress endogenous Gnao1 in murine neurons. Our results will further simplify the study of the pathological mechanism for clinical variants of GNAO1, as well as optimize the testing of gene therapy approaches for GNAO1 encephalopathy in cell models.

[Dynamics of Eukaryotic mRNA Structure during Translation].

Currently, there is no single concept of the optimal spatial structure of mRNA during translation. It is known that many proteins, associated with the 5' end of mRNA, interact with proteins associated with the 3' end. Moreover, this interaction often affects the activity of these proteins. It is possible within the same mRNA molecule only when the mRNA forms a circular structure in which its ends are spatially close. Discovery of such proteins, in the 90s of the 20th century, made it possible to formulate the closed-loop mRNA structure hypothesis, in which it is assumed that the ends of translationally active mRNA are fixed next to each other due to the interaction of proteins and (or) RNA. However, later it was shown that a closed-loop structure is not always necessary for translation. Moreover, some authors have proposed a model according to which the translating mRNA, on the contrary, should be unfolded into a linear structure. Thus, the spatial structure of the translating mRNA does not have to be universal for all mRNA and can change dynamically, which affects its functional activity. In this review, we have summarized a variety of experimental data and concepts on the relationship between the spatial structure of mRNA and its translational activity.

Muscle-Specific Promoters for Gene Therapy.

Many genetic diseases that are responsible for muscular disorders have been described to date. Gene replacement therapy is a state-of-the-art strategy used to treat such diseases. In this approach, the functional copy of a gene is delivered to the affected tissues using viral vectors. There is an urgent need for the design of short, regulatory sequences that would drive a high and robust expression of a therapeutic transgene in skeletal muscles, the diaphragm, and the heart, while exhibiting limited activity in non-target tissues. This review focuses on the development and improvement of muscle-specific promoters based on skeletal muscle α-actin, muscle creatine kinase, and desmin genes, as well as other genes expressed in muscles. The current approaches used to engineer synthetic muscle-specific promoters are described. Other elements of the viral vectors that contribute to tissue-specific expression are also discussed. A special feature of this review is the presence of up-to-date information on the clinical and preclinical trials of gene therapy drug candidates that utilize muscle-specific promoters.

[The Influence of A/G Composition of 3' Stop Codon Contexts on Translation Termination Efficiency in Eukaryotes].

Translation termination is a finishing step of protein biosynthesis. The significant role in this process belongs not only to protein factors of translation termination but also to the nearest nucleotide environment of stop codons. There are numerous descriptions of stop codons readthrough, which is due to specific nucleotide sequences behind them. However, represented data are segmental and don't explain the mechanism of the nucleotide context influence on translation termination. It is well known that stop codon UAA usage is preferential for A/T-rich genes, and UAG, UGA-for G/C-rich genes, which is related to an expression level of these genes. We investigated the connection between a frequency of nucleotides occurrence in 3' area of stop codons in the human genome and their influence on translation termination efficiency. We found that 3' context motif, which is cognate to the sequence of a stop codon, stimulates translation termination. At the same time, the nucleotide composition of 3' sequence that differs from stop codon, decreases translation termination efficiency.

[Expanding the Genetic Code: Unnatural Base Pairs in Biological Systems].

The genetic code is considered to use five nucleic bases (adenine, guanine, cytosine, thymine and uracil), which form two pairs for encoding information in DNA and two pairs for encoding information in RNA. Nevertheless, in recent years several artificial base pairs have been developed in attempts to expand the genetic code. Employment of these additional base pairs increases the information capacity and variety of DNA sequences, and provides a platform for the site-specific, enzymatic incorporation of extra functional components into DNA and RNA. As a result, of the development of such expanded systems, many artificial base pairs have been synthesized and tested under various conditions. Following many stages of enhancement, unnatural base pairs have been modified to eliminate their weak points, qualifying them for specific research needs. Moreover, the first attempts to create a semi-synthetic organism containing DNA with unnatural base pairs seem to have been successful. This further extends the possible applications of these kinds of pairs. Herein, we describe the most significant qualities of unnatural base pairs and their actual applications.

[Genetic composition of the short-stem rye populations (Secale cereale L.) for secaline genes].

Genetic composition for secaline genes was studied in four short stem rye populations (Bolgarskaya korotkostebelnaya, Gnom 1, Gnom 2, and Gnom 3) using the methods of classical hybridological analysis and population genetics. Eight, three, and four alleles of the genes Sec-3, Sec-2, Sec-1 correspondingly have been revealed in the studied populations. The frequencies of the specific alleles in each population were determined. The allele transmission to the next generation is restricted through the linkage of the secaline genes with the genes of self-incompatibility, S1 and Z, located on the same chromosomes as the secaline genes.

[Genetic analysis of oat (Avena sativa L.) by joint scaling test]. / Geneticheskii analiz ovsa posevnogo (Avena sativa L.) s pomoshch'iu testa sovmestnogo shkalirovaniia.

Comparing the results of genetic analysis of oat varieties with the method of diallel analysis of F1 hybrids and with the joint scaling test the spheres of optimal application of both methods were determined. Quantitative estimation of genetic parameters forming the phenotypic averages in scaling test develops the necessary base for involvement of marker genes for qualitative characters in search of QTLs controlling the traits with continuous variation. The crosses being the most suitable for further investigation with the aim to identify and to allocate the main genes of quantitative traits are indicated.

[Effect of restoration of coronary blood flow on dynamics of myocardial perfusion and function in patients with acute myocardial infarction. Data of gated technetium-99m-sestamibi SPECT]. / Vliianie vosstanovleniia koronarnogo krovotoka na dinamiku perfuzii i funktsii miokarda u bol'nykh ostrym infarktom po dannym sinkhronizirovannoi tomografii s 99mTs-MIBI.

Effects of coronary blood flow restoration on dynamics of myocardial perfusion and function were studied in 50 patients with acute myocardial infarction during 6-month follow-up. Gated SPECT with 99mTc-MIBI (G-SPECT) was performed before onset of reperfusion procedures, in 24 hours, 10 days, 1 and 6 months. In patients with successful reperfusion (n=36) perfusion defect (PD) size decreased by 6.78+/-0.97% (p<0.001) and its severity by 221.3+/-85 std (p<0.001) in 24 hours. In 10 days PD size decreased by 3.9+/-0.88% (r<0.05) and severity by 149+/-39 std (p<0.05), whereas in 1 month PD size decreased by 2,18+/-1,4% (p<0.05), PD severity by 146.3+/-67 std (p<0.05). Successful reperfusion was associated with improvement of contractility: left ventricular ejection fraction increased by 3% (p<0.05) on day 10 of the disease. In patients with failed reperfusion PD size and severity decreased in 10 days by 4.8+/-4.2%, p<0.05, and 276+/-75 std, p<0.05, respectively, in 6 months PD size decreased by 3.1+/-0.9%, p<0.05, left ventricular ejection fraction did not change during follow-up. We conclude that successful reperfusion therapy promotes restoration of myocardial perfusion and improvement of its contractility.

[Effects of intervarietal genetic heterogeneity in oats by results of F1 diallele analysis]. / Vyiavlenie mezhsortovoi geneticheskoi geterogennosti ovsa po rezul'tatam diallel'nogo analiza F1.

Genetic analysis of seven oat varieties for eight traits which are the yield components was performed when studying the varieties and the F1 hybrids from their diallel crosses. Involvement of epistatic genes in genetic control of all the traits excluding the panicle density was established. By analysing the diallel tables with excluded data from the varieties with epistatic genes, it was revealed that hyperdominance is inherent to genetic control of the majority of traits studied. The dominant gene action exceeds the additive gene action. The genes "u" increasing the phenotypic displaying of the trait are always dominant and their frequency in the variety set under consideration is higher than the frequency of the genes "v" for all traits studied.