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.

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.

Obesity is a significant metabolic disorder associated with excessive fat accumulation and insulin resistance. In this study, we explored a gene therapy approach to treat obesity in agouti mice using adeno-associated viruses (AAVs) carrying PRDM16, FoxP4, or Follistatin (FST) genes, which are known to promote the browning of white adipose tissue. Mice treated with AAVs encoding PRDM16, FoxP4, or FST genes showed a reduction in body weight (10–14%) within the first three weeks after administration, compared to the control groups. A lipidomic analysis of the adipose tissue revealed a dramatic reduction in triacylglycerol (TAG) species with low carbon numbers (40–54 acyl carbons) in treated mice.

Adeno-associated viruses (AAV) are widely used as viral vectors for gene delivery in mammalian cells. We focused on the efficacy of transduction of AAV 2/5, 2/6, 2/8 and 2/9 expressing GFP in 3T3-L1 murine preadipocyte cells by live imaging microscopy using IncuCyte S3 and flow cytom-etry. Three transduction modes were assessed: AAV transduction in 3T3-L1 preadipocyte cells with or without further differentiation into mature adipocyte-like cells and injection of AAVs in differ-entiated adipocyte-like cells. 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 effi-ciencies 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 inter-scapular adipose tissue. 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 AAV8 and AAV9 for gene delivery to adipocytes.

Obesity is a significant metabolic disorder associated with excessive fat accumu-lation and insulin resistance. In this study, we explored a gene therapy approach to treat obesity in agouti mice using adeno-associated viruses (AAVs) carrying PRDM16, FoxP4, or Follistatin (FST) genes, which are known to promote the browning of white adipose tissue. Mice treated with AAVs encoding PRDM16, FoxP4, or FST genes showed a reduction in body weight (9-12%) within the first three weeks after admin-istration, compared to control groups. Lipidomic analysis of adipose tissue revealed a dramatic reduction in triacylglycerol (TAG) species with low carbon number (40-54 acyl carbones) in treated mice.

Obesity is a global health threat, which continues to rise in prevalence worldwide, driving increases in related chronic diseases such as type 2 diabetes and cardiovascular conditions. While the sheer scale of the crisis affects over a billion people worldwide, the challenge is compounded for a distinct population: patients with genetically inherent obesity. Gene therapy targeting adipose tissue offers promise for treating obesity, but it requires development of tissue-targeting methods to minimize off-target effects and ensure safety. Our study presents two strategies to restrict transgene expression specifically to adipocytes: engineered adipose-specific promoters and optogenetic regulation. We designed truncated promoter fragments from murine Ucp1, Adipoq, and Fabp4 genes and evaluated their activity during 3T3-L1 preadipocyte differentiation. While all adipocyte-specific promoters exhibited substantially lower activity than the constitutive Cytomegalovirus promoter (10- to 100-fold reduction), they displayed distinct temporal activation patterns: Ucp1 peaked during active differentiation, whereas Adipoq was predominantly active in mature adipocytes. In vivo validation using AAV6/8 vectors confirmed adipose expression of the fluorescent reporter Katushka2S under the Ucp1 promoter in murine interscapular fat. Bioinformatic analysis (MAST/MCAST) of six adipocyte-enriched genes (Fabp4, Adipoq, Ucp1, Lep, Cidec, Sfrp5) identified high-density transcription factor binding site clusters that can be used for the development of new promoters for adipose-specific expression. Furthermore, we implemented the iLight optogenetic system to achieve light-dependent reassembly of split FOXP4 and PRDM16 transcription factors, demonstrating tunable Ucp1 activation with minimal basal expression. These strategies—development of synthetic adipose-specific mini-promoters and light-inducible regulation—may serve as a basis for developing precision metabolic medicine as these approaches allow spatiotemporal control of transgene expression.