AAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation

Gene therapies provide treatment options for many diseases, but the safe and long-term control of therapeutic transgene expression remains a primary issue for clinical applications. Here, we develop a muscone-induced transgene system packaged into adeno-associated virus (AAV) vectors (AAVMUSE) based on a G protein-coupled murine olfactory receptor (MOR215-1) and a synthetic cAMP-responsive promoter (PCRE). Upon exposure to the trigger, muscone binds to MOR215-1 and activates the cAMP signaling pathway to initiate transgene expression. AAVMUSE enables remote, muscone dose- and exposure-time-dependent control of luciferase expression in the livers or lungs of mice for at least 20 weeks. Moreover, we apply this AAVMUSE to treat two chronic inflammatory diseases: nonalcoholic fatty liver disease (NAFLD) and allergic asthma, showing that inhalation of muscone—after only one injection of AAVMUSE—can achieve long-term controllable expression of therapeutic proteins (ΔhFGF21 or ΔmIL-4). Our odorant-molecule-controlled system can advance gene-based precision therapies for human diseases.

Supplementary Fig. 1 Optimization of a muscone-induced transgene system (MUSE) in HEK-293T cells.(a) Optimization of the different combinations of plasmids encoding MUSE.In total, 6×10 4 HEK-293T cells were co-transfected with different combinatorial configurations of the plasmids encoding the musconeresponsive receptor expression vector pMOR215-1 (PSV40-MOR215-1-pA), the cAMPresponsive promoter (PCRE) driven reporter expression vector pCK53 (PCRE-SEAP-pA), a truncated version of the receptor-transporting protein vector pRTP1S (PSV40-RTP1S-pA), and an olfactory neuron-specific G protein alpha subunit vector pGαolf (PSV40-Gαolf-pA).Transfected cells were cultivated in a culture medium with or without 10 μM muscone for 48 h, and SEAP production in the culture supernatant was profiled.(b) Optimization of the different ratios of plasmids encoding pMOR215-1, pRTP1S, pGαolf, and pCK53.HEK-293T cells were co-transfected with the plasmids encoding pMOR215-1, pRTP1S, pGαolf, and pCK53 at different ratios (w/w/w/w) and cultivated in culture medium with or without 10 μM muscone for 48 hours.SEAP production in the culture supernatant was profiled.Data are presented as means ± SD; n = 3 biologically independent samples.All plasmids are described in Supplementary Tables 1 and 3. Source data are provided as a Source Data file.
receptor potential (TRP) melastatin 8 (TRPM8)-mediated NFAT signaling pathway.(e, f) Crosstalk analysis between the MUSE-mediated cAMP signaling pathway and the TRPM8-mediated NFAT signaling pathway.HEK-293T cells were co-transfected with plasmids expressing luciferase under the control of the MUSE-mediated cAMP signaling pathway and plasmids encoding SEAP expression under the control of the TRPM8-mediated NFAT signaling pathway.Transfected cells were cultured in the presence or absence of menthol (50 μM) with or without muscone (10 μM).(g) Schematic representations of the MUSE-mediated cAMP signaling pathway and the toll-like receptor 2 (TLR2)-mediated NF-κB signaling pathway.(h, i) Crosstalk analysis between the MUSE-mediated cAMP signaling pathway and the TLR2mediated NF-κB signaling pathway.HEK-293T cells were co-transfected with plasmids expressing luciferase under the control of the MUSE-mediated cAMP signaling pathway and plasmids encoding SEAP expression under the control of the plasmids encoding TLR2-mediated NF-κB signaling pathway.Transfected cells were cultured in the presence or absence of CU-T12-9 agonists (50 μM) with or without muscone (10 μM).SEAP and luciferase expression were profiled 24 hours after induction.Data are presented as means ± SD; n = 3 biologically independent samples.P values were obtained from two-tailed unpaired t-tests.n.s., not significant.All plasmids are described in Supplementary Tables 1 and 3 Data in b are presented as means ± SD; n = 3 biologically independent samples.All plasmids are described in Supplementary Tables 1 and 3. Source data are provided as a Source Data file.Supplementary Fig. 7 The kinetic of AAVMUSE-mediated luciferase expression in vitro.(a) Schematic for the schedule and experimental procedure for the musconecontrolled gene expression.HEK-293T cells (6×10 4 ) transfected with the AAVMUSE system (pWX126, pWX127, pWX158) were cultivated for the indicated periods (0 to 24 hours) in the presence of 0 or 10 μM muscone.Then cells were collected immediately at the indicated time points (X-axis, 0 to 24 hours) and divided into two groups; one (group A) was used to measure luciferase activity (b), and the other one (group B) was collected to extract RNA for qPCR analysis of the luciferase reporter (c).
HEK-293T cells were co-transfected with pWX126/pWX345 and cultivated for the indicated periods (0 to 72 hours) with or without 10 μM muscone.ΔmIL-4 levels in the culture supernatant were quantified at the indicated time points (X-axis, 12 to 72 hours).
Data are presented as means ± SD; n = 3 biologically independent samples.P values were obtained from two-tailed unpaired t-tests.***P < 0.001, ****P < 0.0001.Descriptions of all plasmids, and detailed descriptions of the genetic constructs, are provided in Supplementary Tables 1 and 3. Source data are provided as a Source Data file.
Table S1.Plasmids designed and used in this study.
transduced with control AAVLuc (2 × 10 11 vg).One week after AAV injection, the transduced NAFLD model mice were exposed to nebulized muscone for 4 hours once each week until week 31 or were exposed to a vehicle (a mixture of castor oil and ddH2O).The examined controls included NAFLD model mice transduced with AAVMUSE-ΔhFGF21 and exposed to a vehicle, and NAFLD model mice transduced with AAVLuc with or without exposure to muscone.(b) The fat mass and (c) lean mass were determined by magnetic resonance imaging at 19 weeks after starting the HFFCD diet (purple arrow).(d-g) Indirect calorimetry was performed 20 weeks after starting the HFFCD diet (green arrow) in NAFLD model mice.(d) Volume of O2 (VO2) consumption, normalized to body weight.(e) Area under the curve for O2 consumption.(f) Volume of CO2 (VCO2) expiration, normalized to body weight.(g) Area under the curve for CO2 expiration.The black x-axis line segments represent the 12-h dark phases.(h-o) Plasma levels of AST, ALT, total cholesterol (T-CHO), and triglycerides (TG) were measured at 21 weeks and 31 weeks after starting the HFFCD diet (orange arrows).Data in b-c and e-o are presented as means ± SEM (d-g, n = 4; b-c and h-o, n =5 mice).P values were obtained from two-tailed unpaired t-tests.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Source data are provided as a Source Data file.