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

Wu, Xin; Yu, Yuanhuan; Wang, Meiyan; Dai, Di; Yin, Jianli; Liu, Wenjing; Kong, Deqiang; Tang, Shasha; Meng, Meiyao; Gao, Tian; Zhang, Yuanjin; Zhou, Yang; Guan, Ningzi; Zhao, Shangang; Ye, Haifeng

doi:10.1038/s41467-024-45383-z

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Published: 06 February 2024

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

Nature Communications volume 15, Article number: 1122 (2024) Cite this article

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Abstract

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 (AAV_MUSE) based on a G protein-coupled murine olfactory receptor (MOR215-1) and a synthetic cAMP-responsive promoter (P_CRE). Upon exposure to the trigger, muscone binds to MOR215-1 and activates the cAMP signaling pathway to initiate transgene expression. AAV_MUSE 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 AAV_MUSE to treat two chronic inflammatory diseases: nonalcoholic fatty liver disease (NAFLD) and allergic asthma, showing that inhalation of muscone—after only one injection of AAV_MUSE—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.

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Introduction

Chronic diseases are long-lasting conditions with persistent effects and are a significant challenge for healthcare systems¹. At present, therapeutic protein-based therapies, including antibodies and cytokines, are becoming widespread for treating chronic diseases; challenges with these macromolecular medications include the need for frequent parenteral administration by injections into skin, muscle, or veins, and high-dose treatment, which brings unwanted side effects such as allergic reactions and skin rashes^2,3. There is an urgent need to develop safe and long-term therapeutic strategies for treating chronic diseases^4,5,6.

Owing to low immunogenicity and lack of pathogenicity, recombinant vectors based on adeno-associated viruses (AAVs) are gaining popularity as a platform for gene delivery to treat a variety of human diseases⁷, and they have been successfully applied in both preclinical and clinical settings⁸. Moreover, AAV-based gene therapies can provide long-term therapeutic protein expression, which can obviate the need to manufacture and frequently inject short-lived recombinant proteins⁹. Recently, therapeutic genes have been delivered to specific, desired tissues by manipulating different serotypes and engineering AAV capsids^10,11,12. However, the widespread adoption of AAV-based gene therapy technologies is limited partly by constitutive gene expression, which can lead to toxicity from supraphysiological levels of the therapeutic agent protein(s). For instance, AAV2/9-delivered survival motor neuron overexpression results in sensorimotor toxicity and reverses the initial benefits for spinal muscular atrophy therapy¹³, and AAV-delivered fibroblast growth factor 21 (FGF21) results in bone loss owing to constitutive gene expression at physiological concentrations¹⁴. Accordingly, there is a growing awareness that high and persistent transgene expression levels lead to side effects, and it is necessary to develop AAV vector technologies that enable the induction of gene expression within a suitable therapeutic window^15,16,17.

Towards this goal, various chemically-inducible systems have been incorporated into AAV vectors to control gene expression, for example, upon antibiotic exposure (e.g., tetracycline (Tet)¹⁸ and erythromycin¹⁹). However, these trigger molecules are not suitable for controlling gene expression, because the use of antibiotics may promote antibiotic resistance and cause undesirable effects on animals, including cytotoxicity and tissue invasiveness^20,21. The aroma compound, such as muscone secreted from male musk deer, has attracted interest for many years due to its fascinating and pleasant odor used in perfumes and its physiological effects on humans²². Therefore, muscone is an ideal inducer for inducible systems to achieve needle-free aromatherapy.

Here, we develop a muscone-induced transgene system (MUSE) by taking advantage of a volatile and traceless muscone trigger. We package it into AAV vectors (hereafter referred to as “AAV_MUSE”) to achieve controllable expression of therapeutic genes through inhalation of the muscone trigger, which provides needle-free aromatherapy. The AAV_MUSE system enables remote, muscone dose- and exposure-time-dependent induction of AAV-delivered transgene expression with a negligible background without the stimulus. We show repeated transgene activation that is inducible over 20 weeks after a single AAV administration. We also demonstrate that an AAV_{MUSE-ΔhFGF21} system using the liver tropism serotype AAV2/9 can temporally regulate the production of a therapeutic protein (ΔhFGF21) to treat nonalcoholic fatty liver disease (NAFLD) model mice and show that a MUSE iteration employing a combination of AAV serotypes (AAV2/6 and AAV2/lung) can control production of the therapeutic protein (ΔmIL-4) to treat allergic asthma model mice (Fig. 1a, b). Our study thus illustrates that AAV_MUSE is a valuable addition to the toolbox of controlled gene therapies that can improve safety and efficacy and advance gene therapy applications in precision medicine.

**Fig. 1: Schematic showing the mouse experimental design for AAV_MUSE-mediated gene therapy to treat chronic diseases.**

Results

Design and functional validation of MUSE

To generate a muscone-induced transgene system (MUSE), we constitutively expressed the muscone-responsive murine olfactory receptor (MOR215-1) protein²³ and a truncated version of receptor-transporting protein 1 (RTP1S), which is known to facilitate MOR215-1 trafficking to the cell surface membrane²⁴. When exposed to muscone, muscone binds to the G-protein-coupled receptor MOR215-1 to trigger an intracellular surge of the second messenger cAMP via Gα_olf-mediated activation of adenylate cyclase (AC)²⁵. cAMP can then bind to the regulatory subunits of protein kinase A (PKA), the catalytic subunits of which translocate into the nucleus, where they phosphorylate the cAMP-responsive binding protein 1 (CREB1), which binds to a synthetic cAMP-responsive promoter (P_CRE) to initiate transgene expression (Fig. 1c).

To test whether MUSE can induce gene expression, we transfected human embryonic kidney cells (HEK-293T) with different combinations of plasmids encoding the muscone-responsive vector pMOR215-1, the cAMP-responsive promoter (P_CRE) driven reporter gene vector pCK53, a truncated version of the receptor-transporting protein vector pRTP1S, and an olfactory neuron-specific G protein alpha subunit vector pGα_olf. The combination containing pMOR215-1, pRTP1S, pGα_olf, and pCK53 had high induction (28-fold induction) of secreted human placental alkaline phosphatase (SEAP) expression in the presence of muscone in the culture medium (Supplementary Fig. 1a). To further improve reporter induction efficiency, we tested different ratios of plasmids encoding MUSE and found that a combination of pMOR215-1, pRTP1S, pGα_olf, and pCK53 at a 5:1:1:1 ratio (w/w/w/w) showed the highest induction (63-fold) of SEAP expression in the presence of muscone in the culture medium (Supplementary Fig. 1b). Importantly, MOR215-1 could only be activated by muscone structural analogs (Supplementary Fig. 2) and was completely insensitive to the endogenously produced molecules that might cross-activate the cAMP signals (Supplementary Fig. 3). We also confirmed that MUSE was functional in additional mammalian cell lines (e.g., HeLa, hMSC-TERT, and Hana 3 A), suggesting its broad applicability (Fig. 2a).

**Fig. 2: Characterization of muscone-induced transgene system (MUSE) performance.**

To assess the possible cytotoxicity of muscone on cells, HEK-293T cells transfected with a constitutive SEAP expression vector (pSEAP2-control) were exposed to different concentrations of muscone (0–80 μM). SEAP expression was not reduced, indicating that muscone exposure did not influence gene expression capacity (Supplementary Fig. 4a). Moreover, according to the cell viability assay, we did not observe any apparent toxicity for cells containing MUSE exposed to 0–80 μM muscone (Supplementary Fig. 4b), indicating that the MUSE components and 10 μM muscone are well tolerated by cells. Importantly, we found no apparent crosstalk between the MUSE-mediated cAMP signaling pathway and three other signaling pathways, including mitogen-activated protein kinase (MAPK)-mediated protein kinase (IRS-1-Ras-MAPK) pathway through the activation of insulin receptor (IR)²⁶, intracellular calcium-dependent nuclear factor of activated T cells (NFAT) pathway through the activation of transient receptor potential (TRP) melastatin 8 (TRPM8)²⁷, and proinflammatory nuclear factor NF-κB activation in response to toll-like receptor 2 (TLR2) signaling²⁸ (Supplementary Fig. 5a–i).

To evaluate MUSE-mediated SEAP expression kinetics, HEK-293T cells were co-transfected with MUSE-encoding plasmids and exposed to different concentrations of muscone (0–80 μM); SEAP expression was profiled every 24 h. SEAP expression was induced in a dose-dependent manner upon adjusting the muscone concentration (ranging from 0.1 to 10 μM) and achieved saturation above 10 μM muscone (Fig. 2b). HEK-293T cells co-transfected with MUSE-encoding plasmids were exposed to 10 μM muscone for various time periods; the anticipated time-dependent SEAP expression was detected (Fig. 2c). To characterize the remote responsiveness of MUSE-engineered cells to muscone, 10 μM muscone was placed at the upper left corner well of a 96-well plate; this induced distance-dependent SEAP expression in MUSE-engineered cells that were seeded throughout the other wells of the plate (Fig. 2d). Thus, our designed MUSE enabled safe and remote control of transgene expression in a muscone dose- and time-dependent manner in mammalian cells.

AAV_MUSE-mediated transgene expression in mouse livers

Before transducing mice with MUSE using AAV2/9 delivery (AAV_MUSE), we developed a relatively simple system comprising a few constructs to facilitate in vivo delivery based on a single AAV vector that concatenated the constructs for RTP1S and Gα_olf; or MOR215-1 and RTP1S; or MOR215-1 and Gα_olf. We eventually obtained an optimized concatenated Gα_olf and RTP1S vector pWX127 (ITR-P_SV40-Gα_olf-P2A-RTP1S-pA-ITR). We found that combining the muscone-responsive vector pWX126 (ITR-P_SV40-MOR215-1-pA-ITR), the concatenated Gα_olf and RTP1S vector pWX127, and the reporter vector pWX158 (ITR-P_CRE-luciferase-P2A-EGFP-pA-ITR) at a 5:1:1 (w/w/w) ratio resulted in about 67-fold induction of luciferase expression (Supplementary Fig. 6). Further, AAV_MUSE-transfected HEK-293T cells were induced with muscone in a time-dependent manner, and our results indicated that the AAV_MUSE system exhibited fast kinetics, with significant luciferase expression within 2 h (Supplementary Fig. 7).

After validating the AAV_MUSE-mediated reporter gene expression in cells, we tested whether AAV_MUSE induced luciferase reporter expression upon exposure to nebulized-muscone using an ultrasonic nebulizer (Fig. 3a). Then mice were transduced with AAV2/9 vectors²⁹ carrying the AAV_MUSE system components via tail vein injection. At 2 weeks after the AAV injection, AAV_MUSE-transduced mice exposed to nebulized muscone showed high bioluminescence signal intensities in livers measured using an in vivo imaging system as compared to control mice transduced with AAV_MUSE and exposed to vehicle (a mixture of castor oil and ddH₂O) (Supplementary Fig. 8). We also observed higher bioluminescence signal in the isolated livers compared to other organs, indicating that the AAV2/9 efficiently delivered AAV_MUSE system into the liver after intravenous injection (Supplementary Fig. 9). It might be that liver cells are susceptible to AAV2/9 infection, allowing co-transfection of the three vectors in a single liver cell. Moreover, we measured the muscone concentration in the mouse liver, lung, blood, and in the ultrasonic nebulizer by gas chromatography-mass spectrometry (GC-MS). Our results indicated that the concentration in these regions was within the range of the reported EC50 value for MOR215-1³⁰ (Supplementary Fig. 10).

**Fig. 3: AAV_MUSE-mediated luciferase reporter expression in mouse livers.**

To further characterize the kinetics of AAV_MUSE-mediated luciferase expression, AAV_MUSE-transduced mice were exposed to nebulized muscone and bioluminescence imaging was monitored every two hours (Supplementary Fig. 11a). We found that AAV_MUSE-transduced mice exposure to nebulized muscone for 2 h were sufficient to trigger luciferase expression and the maximum luciferase density was observed at 4 h after muscone exposure (Supplementary Fig. 11b, c). Moreover, AAV_MUSE-mediated luciferase expression was muscone dose- and exposure time-dependent in mice (Fig. 3b, c and Supplementary Fig. 12a, b). These results demonstrate that AAV_MUSE can efficiently induce luciferase expression in mice.

We next tested whether AAV_MUSE re-induced luciferase reporter expression upon re-exposure to nebulized muscone in mice. AAV_MUSE-transduced mice were exposed to nebulized muscone (18 mM) for 4 h once every two weeks; control mice were transduced with AAV_MUSE and exposed to vehicle biweekly. Based on weekly monitoring of bioluminescence imaging, we found that repeated muscone nebulization resulted in re-induction of luciferase expression in the livers of AAV_MUSE transduced mice (Fig. 3d and Supplementary Fig. 12c); mice given AAV_MUSE and exposed repeatedly to muscone displayed reproducible induction of luciferase expression for 28 weeks (Fig. 3e, f and Supplementary Fig. 13). However, AAV_MUSE-induced luciferase expression decreased with time, possibly due to rAAVs as non-replicating circularized episomes, gradually leading to the loss of transduced vectors in mitotic cells after AAV transduction⁷. In addition, the introduction of foreign proteins delivered by rAAVs into a mammalian organism may trigger adaptive or innate immune responses, potentially resulting in a reduction of transgene products^10,31,32,33.

Additional analyses from this long-term study to track potential side effects evident in routine blood test and serum biochemistry analyses showed no differences between AAV_MUSE-transduced mice exposed to muscone and control mice (including AAV_MUSE-transduced mice exposed to vehicle or un-transduced wild-type mice) (Supplementary Fig. 14). In addition, liver tissue samples were collected for Gene Set Enrichment Analysis (GSEA) after a total muscone induction period of 20 weeks. GSEA showed that other potential pathways might not be influenced by cAMP generated by the MUSE system in the livers of AAV_MUSE-transduced mice exposed to muscone as compared to control mice (including AAV_MUSE-transduced mice exposed to vehicle or un-transduced wild-type mice) (Supplementary Fig. 15), indicating that intermittent and transient stimulation of the MOR215-1 had not detrimental effects on the endogenous target genes of the cAMP signaling pathway due to transient nature of cAMP signaling activation^34,35. Collectively, these results indicate that AAV_MUSE enables safe, remote, muscone dose- and time-dependent induction of transgene expression, and is re-inducible in mouse livers for up to 28 weeks upon muscone-mediated aromatherapy.

Therapeutic efficacy of AAV_{MUSE-ΔhFGF21} in diet-induced NAFLD model mice

Pursuing the idea of an AAV_MUSE-based gene therapy to treat NAFLD, we chose a synthetic recombinant Fc-FGF21 variant (ΔhFGF21) as the intervention^36,37; this protein has been examined in a clinical Phase II trial designed to reduce hepatic fat in subjects with NAFLD³⁸. To confirm the biological activity of AAV_MUSE-induced ΔhFGF21, we initially engineered a HEK-SEAP™ βKlotho/FGFR cell line co-transfected with three plasmids³⁹: pβKlotho (P_CMV-βKlotho-pA), pTetR-ELK1 (P_CMV-TetR-ELK1-pA), and pMF111 (P_hCMV*-1-SEAP-pA; P_hCMV*-1, O_TetO7-P_hCMVmin) (Supplementary Fig. 16a). When ΔhFGF21 bound to FGFR1 and its coreceptor β-Klotho, the MAPK signaling pathway was activated to phosphorylate TetR-ELK1 (a synthetic hybrid transcription factor), leading to P_hCMV*-1-driven SEAP reporter expression (Supplementary Fig. 16b). Indeed, we observed that AAV_MUSE-induced ΔhFGF21 mediated time-dependent expression of SEAP in the engineered cells (Supplementary Fig. 16c).

After demonstrating the biological activity of ΔhFGF21, we evaluated the therapeutic efficacy of AAV_{MUSE-ΔhFGF21}-mediated gene therapy in diet-induced NAFLD model mice^40,41. Ten weeks after starting a high-fat, high-fructose, high-cholesterol diet (HFFCD), the induced NAFLD model mice were transduced with AAV_{MUSE-ΔhFGF21} comprising the muscone-responsive vector AAV2/9-pWX126 (1 × 10¹¹ viral genomes (vg)), the concatenated Gα_olf and RTP1S vector AAV2/9-pWX127 (5 × 10¹⁰ vg), and the ΔhFGF21 vector AAV2/9-pWX252 (ITR-P_CRE-ΔhFGF21-pA-ITR, 5 × 10¹⁰ vg) via tail vein (Fig. 4a and Supplementary Fig. 17a). After one week, the transduced NAFLD mice were exposed to nebulized muscone for 4 h once weekly until week 31 or were exposed to vehicle (a mixture of castor oil and ddH₂O). The AAV_{MUSE-ΔhFGF21}-transduced NAFLD mice exposed to nebulized muscone (18 mM) exhibited a significant increase in ΔhFGF21 levels compared to vehicle-exposed control mice transduced with AAV_MUSE over 21 weeks (Fig. 4b). Moreover, the AAV_MUSE-induced ΔhFGF21 expression level reached a range of 20–30 ng/ml, which is within the desired therapeutic window.

**Fig. 4: The therapeutic efficacy of AAV_{MUSE-ΔhFGF21}-mediated gene therapy in diet-induced NAFLD model mice.**

At 19 or 29 weeks after starting the HFFCD diet, magnetic resonance imaging showed that AAV_{MUSE-ΔhFGF21}-transduced NAFLD mice had significantly decreased fat mass percentage values and increased lean mass percentage values compared to control groups (including AAV_{MUSE-ΔhFGF21}-transduced NAFLD mice exposed to vehicle, and AAV_Luc-transduced NAFLD mice with or without exposure to muscone) (Fig. 4c, d and Supplementary Fig. 17b, c). To assess the effects of AAV_{MUSE-ΔhFGF21} on metabolism, we placed the mice in indirect calorimetry cages at 20 or 27 weeks after starting the HFFCD diet and found that NAFLD mice transduced with AAV_{MUSE-ΔhFGF21} and exposed to muscone had significantly elevated oxygen consumption and carbon dioxide expiration (Supplementary Fig. 17d–g and Fig. 4e–h) compared to the relevant control mice at both time points. A battery of metabolic tests measuring plasma levels of liver enzymes, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as total cholesterol (T-CHO) and triglyceride (TG) (Supplementary Fig. 17h–o), and an intraperitoneal glucose tolerance test (IPGTT) (Supplementary Fig. 18), consistently showed significant decreases in the AAV_{MUSE-ΔhFGF21}-transduced NAFLD mice exposed to muscone as compared to the relevant control mice. Moreover, the AAV_{MUSE-ΔhFGF21}-transduced NAFLD mice exposed to muscone had significantly reduced body weight compared to the appropriate control mice (Fig. 4i, j).

Next, livers and epididymal white adipose tissue (eWAT) of mice were collected at the pre-determined end-point of 31 weeks. The AAV_{MUSE-ΔhFGF21}-transduced NAFLD mice exposed to muscone displayed significant decreases in the weight of livers and eWAT compared to the relevant control mice (Fig. 4k–n). Hematoxylin and eosin (H&E) and Sirius red staining of liver sections revealed that AAV_{MUSE-ΔhFGF21}-transduced NAFLD mice exposed to muscone exhibited smaller adipose droplets, decreased severity of hepatic steatosis and hepatic fibrosis in the livers as compared to the relevant control mice (Fig. 4o). Taken together, these results confirm that AAV_{MUSE-ΔhFGF21} achieves re-induction of therapeutic protein at an appropriate level for treating NAFLD in diet-induced model mice after only one injection of AAVs.

AAV_MUSE-mediated transgene expression in mouse lungs

To test whether AAV_MUSE enables induction of transgene expression in mouse lungs, we initially used AAV2/lung⁴² to package AAV_MUSE containing three AAV vectors: pWX126 (ITR-P_SV40-MOR215-1-pA-ITR), pWX127 (ITR-P_SV40-Gα_olf-P2A-RTP1S-pA-ITR), and pWX158 (ITR-P_CRE-luciferase-P2A-EGFP-pA-ITR). Upon tail vein injection of varying titers of these vectors to BALB/c mice, no strong luciferase signal was detected in the lung (Supplementary Fig. 19). We speculated that the number of AAV_MUSE component vectors or the capsid of the AAV vectors might have affected gene delivery efficiency.

Seeking to facilitate in vivo delivery, we first simplified AAV_MUSE into two separate AAV vector plasmids: a muscone-responsive vector pWX126 and a concatenated vector for two expression cassettes (pWX322, ITR-P_SV40-RTP1S-pA::P_CRE-SEAP-pA-ITR or pWX325, ITR-P_SV40-RTP1S::pA-SEAP-P_CRE-ITR) (Supplementary Fig. 20a, b). Compared to pWX126/pWX322, the pWX126/pWX325 combination exhibited a higher 21.5-fold induction of SEAP expression and lower background signal. We eventually selected the concatenated construct pWX325 (ITR-P_SV40-RTP1S::pA-SEAP-P_CRE-ITR). A combination of pWX126 and the concatenated vector pWX325 at a 5:1 ratio (w/w) resulted in about 21.5-fold muscone induction of SEAP expression in HEK-293 cells. We also detected about 16.9-fold muscone induction of a firefly luciferase reporter in engineered cells co-transfected with pWX126 and pWX342 (ITR-P_SV40-RTP1S::pA-luciferase-P_CRE-ITR) (Supplementary Fig. 20c). Moreover, we selected AAV serotype combinations for delivering constitutive luciferase (ITR-P_hCMV-luciferase-pA-ITR) to the lung via the tail vein and demonstrated that the combination of AAV2/lung and AAV2/6 resulted in higher bioluminescence signal intensities in lungs compared to either AAV2/6 alone or AAV2/lung alone (Supplementary Fig. 21). We then used a combination of two AAV serotypes for delivery of AAV_MUSE: pWX126 packaged into a AAV2/lung capsid (which was reported to transduce the endothelium of the pulmonary vasculature efficiently) and pWX342 packaged into a AAV2/6 capsid⁴³ (which was reported to transduce both airway and alveolar type II cells efficiently). To test whether AAV_MUSE induced luciferase reporter expression upon exposure to nebulized-muscone in the lung, adult BALB/c mice were transduced with AAV_MUSE comprising AAV2/lung-pWX126 (5 × 10¹¹ vg) and AAV2/6-pWX342 (5 × 10¹¹ vg) via tail vein (Fig. 5a). Two weeks after AAV injection, AAV_MUSE-transduced mice were exposed to nebulized muscone for 4 h once every six weeks or exposed to vehicle (a mixture of castor oil and ddH₂O) (Fig. 5b). We found that AAV_MUSE-transduced mice exposed to nebulized muscone exhibited significantly higher bioluminescence signal intensities (up to 17-fold induction) in lungs compared to vehicle-exposed control mice transduced with AAV_MUSE. Moreover, bioluminescence imaging at different time points showed that the AAV_MUSE-transduced mice exposed to nebulized muscone expressed the luciferase reporter in lungs for up to 20 weeks (Fig. 5c, d). Collectively, these results indicate that the AAV_MUSE enables long-term induction of transgene expression in the lungs when the animal is exposed regularly to the inducer.

**Fig. 5: AAV_MUSE-mediated luciferase reporter expression in mouse lungs.**

Therapeutic efficacy of AAV_MUSE-ΔmIL-4 in allergic asthma model mice

We next developed AAV_MUSE for an in vivo gene therapy application to control the expression of a murine IL-4 mutant protein (ΔmIL-4)⁴⁴, which has been recently reported to protect against allergic asthma⁴⁵. To verify the biological activity of AAV_MUSE-induced ΔmIL-4, we initially engineered HEK-SEAP™ IL-4Rα/IL-13Rα1 cells^46,47 by co-transfecting plasmids including an interleukin-4 receptor α subunit plasmid pIL-4Rα (P_CMV-IL-4Rα-pA), an interleukin-13 receptor α1 subunit plasmid pIL-13Rα1 (P_CMV-IL-13Rα1-pA), a mouse signal transducer and activator of transcription 6 plasmid pSTAT6 (LTR-P_CMV-mSTAT6::P_PGK-puromycin-LTR), and a STAT6-inducible SEAP reporter plasmid pWX326 (P_STAT6-SEAP-pA; P_STAT6, O_STAT6-P_min) (Supplementary Fig. 22a). Consistent with the expected outcomes, when ΔmIL-4 bound to the IL-4Rα/IL-13α1 heterodimeric receptor complex, IL-4/IL-13 signaling was suppressed, which inhibited P_STAT6-driven SEAP reporter expression (Supplementary Fig. 22b). We observed that AAV_MUSE-induced ΔmIL-4 expression was muscone exposure time-dependent in these engineered cells (Supplementary Fig. 22c).

After confirming the biological activity of ΔmIL-4, we evaluated the therapeutic efficacy of AAV_MUSE-ΔmIL-4 mediated gene therapy in ovalbumin (OVA)-induced allergic asthma model mice^48,49. Fourteen days before first sensitization, BALB/c mice were transduced with AAV_MUSE-ΔmIL-4 (containing AAV2/lung-pWX126, 5 × 10¹¹ vg, and AAV2/6-pWX345, 5 × 10¹¹ vg) via tail vein injection; control mice were transduced only with the luciferase reporter AAV_Luc (1 × 10¹² vg). All mice were then sensitized by intraperitoneal (i.p.) injection of 25 µg OVA (Grade V) adjuvanted with aluminum hydroxide (Alum) on days 1 and 7, followed by challenge with aerosol inhalation of 5% OVA (Grade II) for 20 min on days 14, 16, 18, and 20 (Fig. 6a).

**Fig. 6: The therapeutic efficacy of AAV_MUSE-ΔmIL-4 mediated gene therapy in the OVA-induced allergic asthma model mice.**

One day before the OVA challenge, the AAV_MUSE-ΔmIL-4-transduced mice or AAV_Luc-transduced control mice were exposed to nebulized muscone or vehicle for 4 h on days 13, 15, 17, and 19. We found that the AAV_MUSE-ΔmIL-4-transduced group exposed to muscone induced significantly higher ΔmIL-4 levels compared to AAV_MUSE-ΔmIL-4-transduced control mice exposed to vehicle (Fig. 6b). On day 22, lungs and bronchoalveolar lavage fluid (BALF) were collected and flow cytometry analysis showed that AAV_MUSE-ΔmIL-4-transduced mice exposed to muscone had significantly reduced counts of eosinophils (CD11b⁺siglec-F⁺) and T lymphocytes (CD3⁺) in both BALF and lungs as compared to control groups (including AAV_MUSE-ΔmIL-4-transduced allergic asthma model mice exposed to vehicle, AAV_Luc-transduced allergic asthma mice with or without exposure to muscone) (Fig. 6c–f and Supplementary Fig. 23).

Moreover, the levels of Th2-type pro-inflammatory cytokines (IL-4, IL-5, IL-13) and total IgE levels were significantly decreased in the BALF of AAV_MUSE-ΔmIL-4-transduced mice exposed to muscone as compared to the relevant controls (Fig. 6g–j). Additionally, H&E staining of lung sections revealed that AAV_MUSE-ΔmIL-4-transduced mice exposed to muscone exhibited significantly decreased eosinophil infiltration surrounding airways as compared to the relevant controls (Fig. 6k). Together, these results demonstrate that AAV_MUSE-ΔmIL-4 can remotely control ΔmIL-4 to inhibit eosinophil infiltration and pro-inflammatory cytokines and to prevent the development of sensitivity to OVA in allergic asthma mice based on inhalation of muscone.

Discussion

AAV vectors are a leading platform for gene delivery to treat a variety of human diseases⁷. However, clinical experience with multiple AAV-based gene therapies has raised awareness that the supraphysiological levels of transgene expression might induce potential adverse effects¹⁵. Therefore, inducible gene expression systems are attractive approaches for controlling the expression of AAV-delivered genes. Here, we developed AAV_MUSE for remote induction of AAV-delivered transgene expression in a muscone dose- and exposure-time-dependent manner. We demonstrate that AAV_MUSE iterations can be efficiently transduced into mouse livers or lungs, and can induce reporter gene expression for at least 20 weeks after a single AAV administration when mice are exposed repeatedly to nebulized muscone. Importantly, we have also demonstrated that AAV_MUSE-based gene therapy achieved dynamic regulation of therapeutic protein production (ΔhFGF21 in liver and ΔmIL-4 in lung) at physiologically appropriate levels in NAFLD and allergic asthma mice models through one-time intravenous delivery of AAV_MUSE followed by exposure to muscone.

AAV_MUSE is based on a muscone trigger; it employs needle-free aromatherapy⁵⁰, a convenient alternative to injection for chronic conditions such as diabetes, obesity, and chronic kidney disease. Moreover, our AAV_MUSE can be delivered directly to tissues of interest and achieve long-term, controllable production of therapeutic proteins after only one injection of AAV vectors. These aspects are very attractive for therapy applications requiring sustained gene expression compared to cell-based therapies, which require cell collection and ex vivo culture⁵¹. Moreover, some therapeutic cells might be encapsulated inside a semipermeable and immune isolating container to enhance xenogeneic cell survival⁵², making it challenging to achieve long-term gene expression.

MUSE system components can be delivered into various target tissues by engineering the AAV capsid with enhanced tissue tropism, for example, by using directed evolution and through different modes of administration⁵³. It may be possible to deliver MUSE to the brain using the brain tropism serotype AAV-PHP.eB⁵⁴ to control production of a therapeutic protein (e.g., methyl-CpG binding protein 2, MECP2) for treating Rett syndrome (RTT), which could avoid promotion of neurological disorders known to result from aberrant overexpression of MECP2⁵⁵. MUSE could also be used to regulate the production of erythropoietin (EPO) for treating anemia associated with chronic kidney disease⁵⁶ using the engineered muscle tropism serotype MyoAAV after systemic administration⁵⁷, which could avoid the induction of high plasma EPO levels linked to elevated risk for cardiovascular complications⁵⁸. AAV_MUSE could also be transduced into liver tissue to serve as a bio-factory to control the production of various therapeutic proteins based on controlling muscone dose and exposure time compared to traditional drugs that are static and cannot be adjusted, including thymic stromal lymphopoietin (TSLP)⁵⁹ for treating obesity and FGF21 for treating diabetes⁶⁰.

Despite our successful demonstrations of treating chronic diseases using AAV_MUSE-based gene therapies, some barriers must be overcome before chemically inducible systems can be widely accepted for a broad range of diseases, such as the potential cytotoxicity from the chemical inducers that affect cell numbers and colony formation⁶¹, and the stability and metabolism of the chemical inducer that affects the duration and consistency of gene expression or diffusing freely in vivo⁶². Moreover, multiple components of our system are an obstacle to broad application. Possible solutions include designing a smaller version of MUSE suitable for packaging within a single AAV by truncating and mutating the AAV_MUSE modules containing RTP1S and MOR215-1, perhaps based on artificial intelligence (AI)-guided protein structure prediction and classification^63,64 or via high-throughput ligand screening⁶⁵. Another idea for the efficient in vivo delivery of MUSE is using engineered virus-like particle (eVLP)⁶⁶, which hold great promise as vehicles for targeted therapeutic macromolecule delivery and represent an improvement over AAV or plasmid delivery. Further, for the tissue specific delivery of our AAV_MUSE system to livers, it can be resolved by employing liver-specific promoters to drive MOR215 and utilizing engineered tissue-specific AAV capsids through directed evolution and computer-guided design⁷. We anticipate that our designed AAV_MUSE system for controlling therapeutic gene expression will be a valuable addition to gene-based therapeutics and can help develop more effective and safer personalized disease treatments.

Methods

Ethical statement

This study did not involve any human data. Experiments involving animals were performed according to a protocol approved by the East China Normal University (ECNU) Animal Care and Use Committee and in accordance with the Ministry of Science and Technology of the People’s Republic of China on Animal Care guidelines. The protocols (protocol ID: m20200213, m20210114) were approved by the ECNU Animal Care and Use Committee. All animals were euthanized after the termination of the experiments.

Animals

Female BALB/c mice (8-week-old) and male C57BL/6 mice (8-week-old) were obtained from the ECNU (East China Normal University) Laboratory Animal Center. The mice were housed at the ECNU Animal Care in 12/12-h light/dark cycles at 22 ± 2 °C and the humidity from 40% to 60%. The 3–5 mice were housed per cage in individually ventilated cages, with ad libitum access to food and water. The mouse gender was taken into account in the study design. For the NAFLD model, male C57BL/6 mice (8-week-old) are commonly chosen to construct the NAFLD model due to their susceptibility to diet-induced obesity and metabolic complications⁶⁷. For the allergic asthma model, female BALB/c mice (8-week-old) are selected to construct the allergic asthma model because female mice have been reported to develop a more pronounced type of allergic airway inflammation than male mice after the OVA challenge⁴⁸. The corresponding figure legends state the sex, age, strain, and number of mice used for each experiment. At the end of the study, mice were euthanized by carbon dioxide exposure.

Plasmid construction

Plasmids constructed and used in this study are provided in Supplementary Table 1. The detailed DNA sequences for MUSE components are provided in Supplementary Table 3. Some plasmids were constructed using ClonExpress Ultra One Step Cloning kit (C116, Vazyme Biotech Co., Ltd) according to the manufacturer’s instructions. All genetic components were confirmed by Sanger sequencing (Shanghai Saiheng Biotechnology). The fragments for ΔhFGF21 [the fusion protein of hFc and hFGF21 (GenBank accession number NC_000019.10) with three mutations (L98R, P171G, and A180E)] and ΔmIL-4 [IL-4 (GenBank accession number NC_000077.7) with two mutations (R116D, Y119D)] were chemically synthesized by the Genewiz company.

Cell culture and transfection

Human embryonic kidney cells (HEK-293T, CRL-11268, ATCC); human cervical adenocarcinoma cells (HeLa, CCL-2, ATCC); HEK-293-derived Hana3A cells engineered for constitutive expression of RTP1, RTP2, REEP1, and G_αoλϕ⁶⁸; and telomerase-immortalized human mesenchymal stem cells (hMSC-TERT, SCRC-4000, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, catalog no. C11995500BT, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, catalog no. FBSSA500-S, AusGeneX) and 1% (v/v) penicillin/streptomycin solution (catalog no. ST488-1/ST488-2, Beyotime). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ and regularly tested for the absence of Mycoplasma and bacterial contamination.

Cells were transfected with an optimized polyethyleneimine (PEI)-based protocol. Briefly, cells were seeded into a 48-well plate (3 × 10⁴ cells per well) and cultivated overnight to 60–70% confluency at the time of transfection. Cells were subsequently co-transfected with corresponding plasmid mixtures for 6 h with 50 μL of PEI and DNA mixture (PEI and DNA at a mass ratio of 3:1 for HEK-293T, hMSC-TERT, and Hana3A, and at a mass ratio of 5:1 for HeLa; PEI, molecular weight 40,000, stock solution 1 mg/mL in double distilled water, catalog no. 24765, Polysciences). At 6 h after transfection, the culture medium was replaced with fresh medium.

Chemicals

Odorants utilized in this study were purchased from Macklin Biochemical Technology Corporation. Castor oil was purchased from Aladdin. The musk odorants and musk analogs were prepared as 200 mM stock solutions in dimethyl sulfoxide or anhydrous ethanol. Menthol (catalog no. HY-75161), dopamine (catalog no. HY-B0451A), and CU-T12-9 (catalog no. HY-110353) were purchased from MedChemExpress (China) and freshly dissolved in water before each experiment. GLP-1 (catalog no. G3265), atrial natriuretic peptide (ANP) (catalog no. A1663), and human insulin solution (catalog no. I9278) were purchased from Sigma-Aldrich (Buchs, Switzerland).

SEAP reporter assay

The expression of human placental SEAP in a cell culture medium was quantified using a p-nitrophenyl phosphate-based light absorbance time course assay. Briefly, 120 µL of substrate solution [100 µL of 2 × SEAP buffer (pH 9.8) containing 20 mM L-homoarginine hydrochloride (catalog no. A602842, Sangon Biotech), 1 mM MgCl₂ (catalog no. A610328, Sangon Biotech), and 21% (v/v) diethanolamine (catalog no. A600162, Sangon Biotech), and 20 µL of substrate solution containing 120 mM p-nitrophenyl phosphate (catalog no. 333338-18-4, Sangon Biotech)] were added to 80 µL of heat-inactivated (65 °C, 30 min) cell culture supernatant⁶⁹. The time course of absorbance at 405 nm was measured at 37 °C using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments) with Gen5 software (version 2.04).

Luciferase assay

HEK-293T cells (6 × 10⁴) were transfected with the AAV_MUSE system (pWX126, pWX127, pWX158) and cultured for indicated time periods (0 to 24 h) in the presence or absence of 10 μM muscone. Then, cells were collected immediately at the indicated time points, and luciferase activity was measured using the Firefly Luciferase Reporter Gene Assay Kit (catalog no. RG005, Shanghai Beyotime Biotechnology Co., Ltd.). The time course of absorbance at 510 nm was measured at 37 °C using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments) with Gen5 software (version 2.04).

Quantitative real-time PCR

Tissue samples from the left lateral lobe of the liver were harvested, snap-frozen in liquid nitrogen, and stored at −80 °C until use. Total RNA was extracted using an RNAiso Plus kit (catalog no. 9109; Takara Bio), and RNA concentration was measured using NanoDrop OneC (ThermoFisher). A total of 1 μg of RNA was reversely transcribed into cDNA using a HiScript® III RT SuperMix with the genomic DNA Eraser (catalog no. R323, Vazyme). Real-time PCR was performed on a Real-Time PCR Instrument (Roche, LightCycler 96, Switzerland) with ChamQ Universal SYBR qPCR Master Mix (catalog no. Q711, Vazyme). The following parameters were used for the PCR: 95 °C for 10 min followed by 40 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min. All samples were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Values and the results were expressed as a relative mRNA amount using the standard 2^-ΔΔCt method. The sequences of primer pairs used in the study are listed in Supplementary Table 2.

RNA-sequencing

Livers were dissected from AAV_MUSE-transduced mice exposed to muscone and control mice (including AAV_MUSE-transduced mice exposed to vehicle or un-transduced wild-type mice), and resuspended in TRIzol reagent (catalog no. 15596026, Invitrogen). Total RNA was extracted and treated with DNase I (Qiagen, Dusseldorf). Library preparation, quality control, and sequencing were performed by Genechem (Shanghai, China). RNA sequencing was performed via the Illumina platform (Illumina Novaseq 6000, CA) based on the mechanism of SBS (sequencing by synthesis). Gene set enrichment analysis (GSEA) was performed on a local tool http://www.broadinstitute.org/gsea/index.jsp. The raw data of RNA-seq was uploaded into the NCBI Sequencing Read Archive (SRA) under accession number PRJNA1011482.

Complete blood count and analysis of liver function

Mice were sacrificed, and whole blood was collected and immediately analyzed for complete blood count using the Sysmex XT-2000iV hematology analyzer (Sysmex). Indicators of hepatic function, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were measured using an automatic biochemical analyzer BX-3010 (Sysmex). All biochemical serum evaluations were performed simultaneously to minimize analytical variability.

AAV production

AAV (serotype 2/9) carrying the muscone-responsive G-protein-coupled receptor MOR215-1 (pWX126, ITR-P_SV40-MOR215-1-pA-ITR), a concatenated Gα_olf and RTP1S expression vector (pWX127, ITR-P_SV40-Gα_olf-P2A-RTP1S-pA-ITR), the inducible luciferase reporter vector (pWX158, ITR-P_CRE-luciferase-P2A-EGFP-pA-ITR), or the inducible ΔhFGF21 expression vector (pWX252, ITR-P_CRE-ΔhFGF21-pA-ITR) were produced by Shanghai Taitool Bioscience. Purified individual AAVs were titered using a quantitative PCR and concentrated in phosphate-buffered saline (PBS, 10 mM, pH 7.4) to 2.15 × 10¹³, 2 × 10¹³, 1.73 × 10¹³, 2.23 × 10¹³ viral genomes (vg)/mL.

AAV (serotype 2/6) carrying pWX342 (ITR-P_SV40-RTP1S::pA-luciferase-P_CRE-ITR) or pWX345 (ITR-P_SV40-RTP1S::pA-ΔmIL-4-P_CRE-ITR) and AAV (serotype 2/lung) carrying pWX126, pWX127, or pWX158 were produced by Shanghai OBiO Technology. Purified individual AAVs were titered using a quantitative PCR and concentrated in PBS to 2 × 10¹³, 2 × 10¹³, 1.8 × 10¹³, 1.4 × 10¹³, or 1.8 × 10¹³ vg/mL.

Mice exposed to nebulized muscone using an ultrasonic nebulizer

Adult female BALB/c mice (8-week-old) or male C57BL/6 mice (8-week-old, East China Normal University) were maintained in an appropriate climatized environment, with controlled conditions of temperature (22 ± 2 °C), humidity (55%), and photoperiod (12 h of light/12 h of dark) for at least 7 days before starting all studies. Mice were transduced with AAV2/9 vectors carrying the AAV_MUSE components via the tail vein. At 2 weeks after AAV injection, the AAV_MUSE-transduced mice were placed into a 285 × 240 × 160 mm chamber and exposed to nebulized muscone [63 μL muscone (catalog no. B21154, Shanghai yuanye Bio-Technology) dissolved in a mixture of 700 μL castor oil (catalog no. C107105, Aladdin) and 3.5 mL ddH₂O] using an ultrasonic nebulizer (SA703, Jiangsu SANS Biological Technology) for different time periods (0–4 h). Four hours after muscone inhalation, bioluminescence images of the mice were obtained using an IVIS Lumina II in vivo imaging system (Perkin Elmer), and analyzed with Living Image software (version 4.3.1).

Instruments and mass spectrometric conditions

The quantitative analysis was performed on an Agilent 7890B gas chromatography system coupled with an Agilent 7000D triple quadrupole mass spectrometer (Agilent Technologies, USA). The processed samples were placed in the vials of the autosampler for injection. The chromatographic column was the DB-5UI capillary column (30 m × 0.25 mm, 0.25 µm). The separation procedure of the GC system was set at the initial temperature of 80 °C held for 1 min, then heated up to 300 °C at the speed of 10 °C/min and held for 5 min. No splitting mode was set. Helium was the carrier gas with a 1.0 mL/min flow rate. The temperature of interface, ion source, and Quadrupole temperature were set at 280 °C, 230 °C, and 150 °C, respectively.

Preparation of standard and sample solutions

The stock solution of muscone (20 μg/mL) was prepared in methanol and diluted with ethyl acetate to produce 0–1000 ng/mL working standard solution used in the linearity experiment. The internal standard (IS) working solution (30 μg/mL) was prepared by diluting Naphthalene stock solutions (2 mg/mL) with ethyl acetate. The Calibration standards were prepared by spiking standard working solution (10 μL) and IS working solution (10 μL) with blank plasma (90 μL) to obtain a serial of muscone concentrations (0, 1, 3, 5, 10, 30, and 100 ng/mL). The sample treatment was a simple liquid-liquid extraction (LLE). The 0.1 mL mouse blood was collected from the orbital venous plexus and transferred to 1.5 mL EDTA-polythene tubes at 4 h after exposure to nebulized muscone. The plasma samples were prepared by spiking 50 μL plasma, 10 μL IS, 50 μL blank plasma with 200 μL neat ethyl acetate. The livers and lungs were dissected from mice 4 h after exposure to nebulized muscone. The liver and lung samples were prepared by spiking 100 μL sample, 10 μL IS with 200 μL neat ethyl acetate. The muscone concentration in the ultrasonic nebulizer’s headspace was analyzed after diluting by 1000 times, and then 100 μL diluted muscone was added with 10 μL IS and 200 μL ethyl acetate. Then, the mixture solution was vortexed for 1.0 min and centrifuged at 13,000 × g at 4 °C for 10 min. The supernatant was transferred into an injection vial for analysis. All the prepared solutions were stored at −20 °C until analysis. 1 μL supernatant was subjected to GC-MS/MS system for analysis.

AAV_MUSE-mediated luciferase expression in mouse livers

Wild-type BALB/c mice (8-weeks-old) were transduced with AAV_MUSE containing the muscone-responsive vector AAV2/9-pWX126 (3 × 10¹¹ vg), the concatenated Gα_olf and RTP1S expression vector AAV2/9-pWX127 (1.5 × 10¹¹ vg), and the inducible reporter expression vector AAV2/9-pWX158 (ITR-P_CRE-luciferase-P2A-EGFP-pA-ITR, 1.5 × 10¹¹ vg) via tail vein. Two weeks after AAV injection, the AAV_MUSE-transduced mice were exposed to nebulized muscone using a nebulizer for different time periods (0–4 h) and different muscone concentrations (0–18 mM); control mice were transduced with AAV_MUSE and exposed to vehicle (a mixture of castor oil and ddH₂O). Four hours after muscone inhalation, each mouse was intraperitoneally injected with luciferin (150 mg kg^–1; catalog no. luc001, Shanghai Sciencelight Biology Science & Technology) under anesthesia. Five minutes after luciferin injection, bioluminescence images of the mice were obtained using an IVIS Lumina II in vivo imaging system (Perkin Elmer), and analyzed with Living Image software (version 4.3.1).

AAV_MUSE-mediated ΔhFGF21 production in diet-induced NAFLD model mice

Eight-week-old male C57BL/6 mice were fed a high-fat, high-fructose, high-cholesterol diet (HFFCD, 40% kcal fat, 20% kcal fructose, and 2% cholesterol; catalog no. D09100301, Research Diet) to induce non-alcoholic fatty liver disease (NAFLD)^40,41. At 10 weeks after starting the HFFCD diet, NAFLD model mice were transduced with AAV_{MUSE-ΔhFGF21} containing the muscone-responsive receptor expression vector AAV2/9-pWX126 (1 × 10¹¹ vg), the concatenated Gα_olf and RTP1S expression vector AAV2/9-pWX127 (5 × 10¹⁰ vg), and the inducible ΔhFGF21 expression vector AAV2/9-pWX252 (ITR-P_CRE-ΔhFGF21-pA-ITR, 5 × 10¹⁰ vg), or transduced with firefly luciferase control vector AAV_Luc (2 × 10¹¹ vg; ITR-P_CBh-mCherry-P2A-luciferase-pA-ITR) via tail vein. One week after AAV injection, the transduced NAFLD model mice were exposed to nebulized muscone or vehicle (a mixture of castor oil and ddH₂O) for 4 h once each week until week 31. The examined controls included NAFLD model mice transduced with AAV_{MUSE-ΔhFGF21} and exposed to a vehicle, as well as NAFLD model mice transduced with AAV_Luc with or without exposure to muscone. All groups of mice were maintained on the HFFCD diet during the study. After 4 h of exposure to muscone, blood samples were collected from mice orbits and transferred to ethylenediaminetetraacetic acid (EDTA) coated mini vacutainer tubes (catalog no. 36597499, BD). After 10 minutes of incubation, samples were centrifuged at 1000 × g for 10 min at 4 °C to separate plasma. ΔhFGF21 levels in mouse plasma were quantified using a human FGF21 ELISA kit (EK1151, MultiSciences Biotech) every 2 weeks from 10 to 31 weeks after starting the HFFCD diet. Body weights were monitored weekly for 31 weeks, and magnetic resonance imaging was performed to measure the fat mass and lean mass at 19 or 29 weeks after starting the HFFCD diet using AccuFat nuclear magnetic resonance (NMR) (AccuFat-1050, MAG-MED).

Measurement of triglycerides (TG) and total cholesterol (T-CHO) levels in mice plasma

At 21 or 31 weeks after starting the HFFCD diet, mouse peripheral blood samples were collected from mice orbits and transferred to EDTA-coated mini vacutainer tubes (catalog no. 36597499, BD). After 10 min of incubation, blood samples were centrifuged at 1000 × g for 10 min at 4 °C. Plasma was separated and stored at −80 °C for further analysis. The levels of triglycerides (TG) and total cholesterol (T-CHO) in mouse plasma were measured using commercial kits [632-50991 (TG) and 294-65801 (T-CHO), Wako] according to the manufacturer’s instructions, and tests were performed with the Synergy H1 hybrid multimode microplate reader (BioTek Instruments) with Gen5 software (version 2.04).

Intraperitoneal glucose tolerance test (IPGTT) in NAFLD model mice

At 22 weeks after starting the HFFCD diet, all mice were fasted for 16 h and received an intraperitoneal injection of D-glucose (1.5 g/kg body weight) dissolved in PBS. The glycemic profile of each animal was monitored via tail vein blood samples at 0, 30, 60, 90, and 120 min after D-glucose administration using a Contour Glucometer (Exactive Easy III, MicroTech Medical). The trapezoidal rule determined the area under the curve (AUC) for IPGTT.

Indirect calorimetry tests

At 20 or 27 weeks after starting the HFFCD diet, all mice were individually housed in metabolic cages of a Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments, Columbus) at 24 °C–26 °C. After a 24-h acclimation period, animals were monitored for 48 h to determine the calorimetric parameters for VO₂, VCO₂, and body weight. All mice were exposed to a daily 12-h light phase (07:00–19:00) and a 12-h dark phase (20:00–06:00).

Ovalbumin (OVA)-induced allergic asthma model mice

To establish an OVA-induced allergic asthma mouse model, each female BALB/c mouse was sensitized by intraperitoneal injection of 25 µg OVA (Grade V, catalog no. A5503, Sigma-Aldrich) emulsified with 2 mg aluminum hydroxide (alum adjuvant, catalog no. 77161, Thermo Fisher Scientific) in 100 µL PBS on days 1 and 7. Mice were then challenged with nebulized 5% OVA [500 mg OVA (Grade II, catalog no. A5253, Sigma-Aldrich) dissolved in 10 mL PBS] for 20 min once at days 14, 16, 18, and 20. Control mice were similarly sensitized and challenged with PBS instead of OVA. To verify that the allergic asthma disease model was successfully established, bronchoalveolar lavage fluid (BALF) and lung tissues were harvested 48 h after the final challenge to assess eosinophil recruitment. Levels of IL-4, IL-5, and IL-13 in BALF were determined using corresponding ELISA kits according to the manufacturer’s instructions.

AAV_MUSE-mediated ΔmIL-4 production in OVA-induced allergic asthma model mice

Fourteen days before the first sensitization (day -14), female BALB/c mice (8-week-old) were transduced with AAV_MUSE-ΔmIL-4 containing AAV2/lung-pWX126 (5 × 10¹¹ vg) and AAV2/6-pWX345 (5 × 10¹¹ vg) via tail vein injection; control mice were transduced only with the luciferase reporter AAV_Luc (1 × 10¹² vg). All mice were then sensitized by intraperitoneal injection of 25 µg OVA (Grade V) adjuvanted with aluminum hydroxide (Alum) on days 1 and 7, followed by challenge with aerosol inhalation of 5% OVA (Grade II) for 20 min once on days 14, 16, 18, and 20. AAV_MUSE-ΔmIL-4-transduced mice or AAV_Luc-transduced control mice were exposed to nebulized muscone or vehicle for 4 h on days 13, 15, 17, and 19. On day 22, mice were euthanized with carbon dioxide asphyxiation, and lungs and BALF were collected for further analysis.

Bronchoalveolar lavage procedure

Mice were euthanized with carbon dioxide asphyxiation, and a small semi-incision of the trachea was made using scissors, allowing a 24-G lavage tube to pass into the trachea. The tube and trachea were stabilized by attaching them with a cotton thread, then a 1-mL syringe was placed into the 24-G lavage tube, and 0.5 mL sterile 1×PBS loaded in the syringe was injected into the lung, which was repeated three times. BALF was pooled and centrifuged for 5 min at 300 × g at 4 °C to pellet the cells. The supernatant was separated and stored at -80 °C for further analysis of cytokines, and the cell pellets were resuspended in 300 μL RPMI 1640 medium for further flow cytometry analysis.

Isolation of lung tissue cells

Mice were euthanized with carbon dioxide asphyxiation, and lung tissues were collected, cut into small pieces, and enzymatically digested in 5 mL solution containing 250 U/mL collagenase VIII (catalog no. C2139, Sigma-Aldrich) and 75 µg/mL DNase I (catalog no. DN25, Sigma-Aldrich) for 60 min at 37 °C with mild shaking. Lung cell suspensions were passed through a 70 μm cell strainer (strong nylon mesh with 70-micron pores, catalog no. 93070, Thermo Fisher Scientific) and centrifuged at 300 × g for 5 min. Pelleted cells were resuspended in 4 mL of 30% Percoll® (catalog no. 17089109-1 EA, Cytiva), and 2.5 mL of 70% Percoll® was added to the bottom. Subsequently, the samples were centrifuged at 600 × g with acceleration (ACE) = 5, deceleration (DCE) = 1 for 20 min, and the intermediate liquid containing the lung tissue cells were washed using 9 mL 1× PBS and then centrifuged at 800 × g for 20 min to remove the supernatants. Finally, the cell pellets were resuspended in 200 µL RPMI 1640 medium (catalog no. 11875093, Gibco) on ice for flow cytometry analysis.

Flow cytometry analysis of eosinophils and lymphocytes in BALF and lung tissues

To quantify the counts of eosinophils, cells from BALF or lung tissues were resuspended in 100 µL RPMI 1640 medium containing BV510 Live/Dead (catalog no. 423101, Biolegend), Alexa Fluor®-700 anti-mouse CD45 (catalog no. 103127, clone 30-F11, Biolegend), KIRAVIA Blue 520^TM anti-mouse F4/80 (catalog no. 123161, clone BM8, Biolegend), Brilliant Violet 421^TM anti-mouse CD11c (catalog no. 117329, clone N418, Biolegend), APC/Cyanine7-anti-mouse CD11b (catalog no. 101226, clone M1/70, Biolegend), and PE anti-mouse CD170 (Siglec-F) (catalog no. 155505, clone S17007L, Biolegend) on ice for 30 minutes. To quantify the counts of T lymphocytes, cells from BALF or lung tissues were resuspended in 100 µL RPMI 1640 medium containing BV510 Live/Dead (catalog no. 423101, Biolegend), FITC anti-mouse CD3ε (catalog no.100306, clone 145-2C11, Biolegend) on ice for 30 min. All cells were washed with PBS and were measured on a BD LSRFortessa^TM Flow Cytometer (BD Biosciences). All antibodies were purchased from Biolegend and were diluted at 1:200. A minimum of 5000 events per plot were collected and analyzed using FlowJo V10 software. The numbers presented in the flow cytometry analysis images are percentage-based. All antibodies were validated for the specified application by respective manufacturer.

Enzyme-linked immunosorbent assay (ELISA)

Levels of ∆hFGF21 in mouse plasma were quantified using a human FGF21 ELISA kit (EK1151, MultiSciences Biotech). Levels of IL-4, IL-5, or IL-13 in mouse BALF were quantified using a mouse IL-4 ELISA kit (abs520003, Absin), a mouse IL-5 High Sensitivity ELISA kit (EK205HS, MultiSciences Biotech), or a mouse IL-13 ELISA kit (KMC2221, Invitrogen), respectively. The levels of total IgE were quantified using a mouse IgE ELISA kit (1218202, Biosci). All ELISA assays were conducted according to the manufacturer’s instructions.

Hematoxylin and eosin (H&E) staining of liver and lung tissues

Mice were sacrificed by carbon dioxide asphyxiation, and samples from liver and lung tissue were collected and fixed in 4% paraformaldehyde (catalog no. G1101, Servicebio) overnight at room temperature. The fixed samples were gently dehydrated by immersing in a graded series of alcohol solutions, cleaned in xylene, embedded in paraffin, and then sliced into 4 µm-thick tissue sections with a rotary microtome (Leica RM2235, Manual Rotary Microtome). These sections were stained with the H&E Staining Kit (catalog no. G1005, Servicebio) according to the manufacturer’s instructions and were observed using an upright microscope (BX53, Olympus) equipped with an Olympus digital camera.

Sirius red staining of liver tissues

Liver tissues were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm sections. Liver sections were stained with 0.05% Sirius red solution (G1018, Servicebio) for 8 min, dehydrated quickly with anhydrous ethanol, cleaned in xylene for 5 min, and sealed with neutral gum. Representative Sirius red staining of liver sections was observed using an upright microscope (BX53, Olympus) equipped with an Olympus digital camera.

Statistical analysis

All in vitro data are expressed as the mean ± SD of three independent biological replicates and are described separately in the figure legends. For the animal experiments, each treatment group consisted of randomly selected mice (n = 3 to 5), and the results are expressed as means ± SEM. Neither animals nor samples were excluded from the study. Comparisons between the two groups were performed using two-tailed unpaired t tests. One-way ANOVA was used to evaluate differences between multiple groups with a single intervention, followed by Dunnett’s post hoc test. Statistical analyses were performed using GraphPad Prism software (version 8), and differences were considered statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n and P values are described in the figures or figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data associated with this study are present in the paper or the Supplementary Information. Source data are provided as a Source Data file with this paper. The raw data of RNA-seq was uploaded into the NCBI Sequencing Read Archive (SRA) under accession number PRJNA1011482. The remaining data are available within the Article, Supplementary Information or Source Data file. All genetic components related to this paper are available with a material transfer agreement and can be requested from H.Y. (hfye@bio.ecnu.edu.cn). Source data are provided with this paper.

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Acknowledgements

We thank Prof. Dr. Kazushige Touhara and Prof. Dr. Mika Shirasu (Department of Applied Biological Chemistry, The University of Tokyo) for providing cDNA for pMOR215-1, pRTP1S, and pGα_olf. We thank Hongyi Zang (Shanghai East Hospital, Tongji University School of Medicine) for assistance with animal experiments. This work was financially supported by grants from the National Key R&D Program of China, Synthetic Biology Research (no.2019YFA0904500), the National Natural Science Foundation of China (no. 32250010, and 32261160373), the Science and Technology Commission of Shanghai Municipality (no. 23HC1410100 and 22N31900300), and the Fundamental Research Funds for the Central Universities to H.Y. This work was also partially supported by National Key R&D Program of China (no. 2019YFA0110802), the National Natural Science Foundation of China (NSFC: no. 32171414), the Natural Science Foundation of Shanghai (no. 23ZR1419500), and the Nature Science Foundation of Chongqing, China (no. CSTB2022NSCQ-MSX0461) to M.W. This work was also partially supported by the Young Scientists Fund of the National Natural Science Foundation of China (no. 32301217) to J.Y. and the Young Scientists Fund of the National Natural Science Foundation of China (no. 32300458) to Y.Z. We thank the ECNU Multifunctional Platform for Innovation (011) for supporting the mouse studies and the Instruments Sharing Platform of School of Life Sciences, East China Normal University. We also thank the support from the Chinese Academy of Sciences Youth Interdisciplinary Team.

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These authors contributed equally: Xin Wu, Yuanhuan Yu, Meiyan Wang.

Authors and Affiliations

Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Dongchuan Road 500, Shanghai, 200241, China
Xin Wu, Yuanhuan Yu, Meiyan Wang, Di Dai, Jianli Yin, Wenjing Liu, Deqiang Kong, Meiyao Meng, Tian Gao, Yuanjin Zhang, Yang Zhou, Ningzi Guan & Haifeng Ye
Institute of Medical Technology, Shanxi Medical University, Taiyuan, Shanxi Province, 030001, China
Xin Wu
Chongqing Key Laboratory of Precision Optics, Chongqing Institute of East China Normal University, Chongqing, 401120, China
Meiyan Wang, Jianli Yin & Haifeng Ye
Department of Breast Surgery, Tongji Hospital, School of Medicine, Tongji University, Xincun Road 389, Shanghai, 200065, China
Shasha Tang
Wuhu Hospital, Health Science Center, East China Normal University, Middle Jiuhua Road 263, Wuhu, Anhui, China
Yang Zhou & Haifeng Ye
Division of Endocrinology, Department of Medicine, Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA
Shangang Zhao

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Xin Wu
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Di Dai
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Yang Zhou
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Contributions

H.Y. conceived of the project. H.Y., X.W., Y.Y. and W.L. designed the experiments and analyzed and interpreted the results. H.Y., X.W., Y.Y., M.W., J.Y. and S.Z. wrote the manuscript. X.W., Y.Y., M.W., W.L., J.Y., D.D., D.K., S.T., M.M., T.G., Y.Z., N.G. and Y.Z. performed the experimental work. All authors edited and approved the final manuscript.

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Correspondence to Haifeng Ye.

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Wu, X., Yu, Y., Wang, M. et al. AAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation. Nat Commun 15, 1122 (2024). https://doi.org/10.1038/s41467-024-45383-z

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Received: 18 April 2023
Accepted: 22 January 2024
Published: 06 February 2024
DOI: https://doi.org/10.1038/s41467-024-45383-z

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