Obesity has become a pandemic, as currently more than half a billion people worldwide are obese. The etiology of obesity is multifactorial, and combines a contribution of hereditary and behavioral factors, such as nutritional inadequacy, along with the influences of environment and reduced physical activity. Two types of adipose tissue widely known are white and brown. While white adipose tissue functions predominantly as a key energy storage, brown adipose tissue has a greater mass of mitochondria and expresses the uncoupling protein 1 (UCP1) gene, which allows thermogenesis and rapid catabolism. Even though white and brown adipocytes are of different origin, activation of the brown adipocyte differentiation program in white adipose tissue cells forces them to transdifferentiate into “beige” adipocytes, characterized by thermogenesis and intensive lipolysis. Nowadays, researchers in the field of small molecule medicinal chemistry and gene therapy are making efforts to develop new drugs that effectively overcome insulin resistance and counteract obesity. Here, we discuss various aspects of white-to-beige conversion, adipose tissue catabolic re-activation, and non-shivering thermogenesis.

The level of transduction efficiency of the target retinal cells affects the choice of AAV serotype and the outcome of gene replacement therapy for inherited retinal diseases. This study focused on the tropism and transduction efficiency of AAV2.7m8-, AAV5-, AAV8-, and AAV9-GFP in ARPE-19 and HEK293 cells. Fluorescence intensity was assessed bi-hourly by means of IncuCyte S3 live imaging microscopy. Within 12 h, AAV2.7m8 demonstrated the highest transduction efficiency at four viral concentrations of 1-, 3-, 6-, and 8 × 10⁴ VG/cell in a dose-dependent manner, followed by AAV5 in ARPE-19 and AAV9 in HEK293 cells. The transduction efficiency of AAV2.7m8 at a dose of 6 × 10⁴ VG/cell was 21, 202, and 323 times higher in ARPE-19 cells and 324, 100, and 52 times higher in HEK293 cells compared to AAV5, AAV8, and AAV9, respectively. This trend remained for 4 days at all viral concentrations, as additionally shown by flow cytometry. At a dose of 6 × 10⁴ VG/cell, AAV2.7m8 (97% GFP-positive cells, GFP +) was nearly two and 10 times as efficient as AAV5 (52% GFP+) and AAV9 or AAV8 (both 9%), respectively, in ARPE-19 cells. In HEK293 cells, 95% of AAV2.7m8-, 26% of AAV9-, 17% of AAV8-, and 12% of AAV5-transduced cells were GFP-positive.

Adeno-associated virus (AAV)-based gene therapy has become one of the key directions of modern translational medicine geared towards treatment of hereditary disorders by means of gene replacement. At the moment, about 5,000 different syndromes are associated with mutations in large genes, which presents a great problem due to the AAV packaging capacity of 5 kilobases. The main strategies for overcoming this obstacle were the creation of truncated gene versions, overloading the viral vector, and separate delivery of partial genetic material to restore the whole gene at the level of DNA, RNA, or protein. At present, genome editing via prime editors, most effectively delivered by AAV, relies on the intein pair used to restore the protein complex. The amazing integration speed of intein-based protein trans splicing technology makes it a versatile tool for a variety of applications, albeit not always successful on the first attempt. This study discusses the key points of working with Ssp, Npu, and Ava inteins of the DnaE group, known as the most effective for assembly of large proteins. Using green fluorescent protein (GFP) as a model, we demonstrate that the successful protein assembly requires not only cysteine at position C+1 but also certain aminoacid residues on either side in its immediate environment. Furthermore, the conformation of extein-intein composition, difficult to predict by computer modeling, has an additional effect, as demonstrated by experimental tests of the three split sites optimal in amino acid composition. The NpuDnaE variant demonstrated the highest kinetics of interaction between the N and C parts in the DnaE group of inteins. Optimization of conditions using NpuDnaE intein led to GFP assembly in 80% of transfected HEK293 cells and in 55% of AAV5-transduced cells, as demonstrated by flow cytometry. The efficiency of GFP assembly post-plasmid DNA transfection or AAV transduction of the HEK293 cell line was 15% higher than that of the ARPE19 cell line. We hope that the obtained data will facilitate the development of gene therapies for the treatment of hereditary disorders caused by mutations in large genes.

Background/Objectives: Adeno-associated viruses (AAVs) are widely used as viral vectors for gene delivery in mammalian cells. We focused on the efficacy of the transduction of AAV2/5, 2/6, 2/8 and 2/9 expressing GFP in preadipocyte cells by live imaging microscopy using IncuCyte S3 and flow cytometry. Methods: Three transduction modes in 3T3-L1 preadipocyte cells assessed: AAV transduction in 3T3-L1 preadipocyte cells, transduction with further differentiation into mature adipocyte-like cells and the transduction of differentiated 3T3-L1 adipocytes. For the in vivo study, we injected AAV2/6, AAV2/8 and AAV2/9 in adipose tissue of C57BL6 mice, and the transduction capacity of AAV2/6, along with AAV2/8 and AAV2/9 was evaluated. Results: AAV2/6 demonstrated the highest transduction efficiency in 3T3-L1 preadipocytes, as it was 1.5–2-fold more effective than AAV2/5, and AAV2/8 in the range of viral concentrations from 2 × 10⁴ to 1.6 × 10⁵ VG/cell. AAV2/5 and AAV2/8 showed transduction efficiencies similar to each other. The expression of GFP under the CMV promoter remained stable for up to 20 days. The induction of 3T3-L1 differentiation in three days after AAV transduction did not alter the GFP expression level, and AAV2/6 showed the best transduction efficiency. AAV2/6 demonstrated the ability to transduce mature adipocytes. These results were confirmed by in vivo studies on C57BL6 mice. AAV2/6 had the highest transducing activity on both inguinal and interscapular adipose tissue. Conclusions: Thus, AAV2/6 has demonstrated higher transduction efficacy compared to AAV2/5, AAV2/8 and AAV2/9 both in 3T3-L1 adipocytes and adipose tissue in vivo, which proves its usability along with AAV2/8 and AAV2/9 for gene delivery to adipocytes.

This article delves into the genetic underpinnings of anxiety, indicating that both state and trait anxiety have heritable components. However, there is no consensus on the degree of heritability, and much remains to be understood about the specific genetic variants involved and their mechanisms of action. The study explores the role of the BDNF gene, which is involved in the synthesis and transportation of brain-derived neurotrophic factor protein, and the AMPD1 gene, which facilitates the conversion of inosine monophosphate to adenosine monophosphate, the intracellular precursor for adenosine in the pathophysiology of anxiety. The methodology of this study involved a combination of genetic testing, psychological assessments, and statistical analysis. Participants were recruited from diverse demographic groups to ensure the findings were broadly applicable. DNA samples were collected for genetic testing, and participants completed the STAI questionnaire to measure their state and trait anxiety levels. The genetic data were analyzed to identify associations between variants in the BDNF and AMPD1 genes and levels of anxiety; specifically, the frequency of these variants in participants with high anxiety scores was compared to those with low anxiety scores. The study provided evidence of the association between BDNF variants and levels of trait anxiety and AMPD1 variants with levels of state anxiety, implicating different biological mechanisms underlying these components of anxiety.

Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of adeno-associated virus-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using an adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of the design, production, and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor-interacting protein like-1 (AIPL1) as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate the expression of human AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type AIPL1) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated significant differences in the expression of genes involved in the innate immune response. In contrast, AAV9-AIPL1wt induced the prominent activation of multiple interferon-stimulated genes. The key part of the possible regulatory molecular mechanism is the activation of dsRNA-responsive antiviral oligoadenylate synthetases, and a significant increase in the level of histone coding genes’ transcripts overrepresented in RNA-seq data (i.e., H1, H2A, H2B, H3, and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing, using relevant animal models to develop future therapeutics for LCA4.