Adipose-Targeted Gene Regulation Using Synthetic Promoters and a Light-Switchable Transcription System

Abstract

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.