Control of excess body fat is one of the greatest healthcare challenges of our time (Afshin et al., 2017; WHO, 2023). With the prevalence of obesity rapidly increasing since the 1990s, China now has the highest number of overweight or obese individuals in the world (Chen et al.,2011). Approximately half of the adults and one-fifth of the children in China face the problem of being overweight or obese (Chen et al.,2011). According to the market research conducted by Huaon (Huaon.com, 2023), the size of AOMs in China has been rapidly increasing since 2016, rising from roughly 3 billion RMB in 2016 to more than 19 billion RMB in 2020 (Figure 2a). This number is expected to reach approximately 59 billion RMB in 2023 (Frost & Sullivan.com, 2023). Also, we analyzed the web search index on Baidu, the biggest search engine provider in China, with AOMs as keywords. We found a striking increase in the search frequency on AOMs since 2019, which is consistent with the findings in the market research (Figure 2b).

Globally, the number of obese individuals worldwide has nearly doubled since 1975. In 2016, over 1.9 billion adults aged 18 and above were overweight, with over 650 million of them being obese (WHO,2018). Market research has also shown that the global market size of AOMs has rapidly risen from approximately 18 billion US dollars in 2016 to approximately 44 billion US dollars in 2023. A similar trend could also be observed on Google trend on Glucagon-like peptide-1 receptor agonist (GLP1R agonist), one of the most widely-used AOMs nowadays, where a significant increase in web searches could be observed since 2021 (Figure 2c).

Overall, by looking into the market research and analyzing the online search trends on our own, we have observed a significantly increasing demand for anti-obesity medications worldwide.

Developing AOMs has been a difficult task, with numerous setbacks arising from both technical and societal factors. To understand the major concerns of the general population on different parameters of AOMs, we conducted a questionnaire-based social study to collect how the public weighs the different aspects of the AOMs. Herein, we asked our participants to rank the key parameters of AOMs (Effectiveness of inducing weight loss, Side Effects, Supplemental efforts such as dietary intervention or physical exercise, Price, and Others) by the importance in their own mind (Figure 3a). Among the 2,015 individuals who filled out our questionnaire, we chose 1,253 individuals who showed intentions of weight control using AOMs (Figure 3b). Strikingly, more than 40% of the individuals we approached ranked the efficacy of weight loss as their prioritized concern regarding AOMs (Figure 3c). In the meanwhile, approximately 53% of the individuals marked side effects as the most important feature to consider regarding AOMs.

With these findings, we then looked into how current AOMs perform in regards of safety and effectiveness. In the past, many AOMs have failed after receiving regulatory approval, mainly due to adverse cardiovascular effects, increased risk of suicide, or higher chances of drug dependence and abuse (Venditti et al., 2014). It has also been described in multiple studies that the body weight loss achievable through most registered AOM resides in a relatively narrow range of 3–7% after 6–12 months of treatment (Ricquier & Bouillaud, 2000), which remains unsatisfactory for most individuals we interviewed. To understand the conditions in a more straightforward manner, we interviewed Dr. Jingjing Zhang, a clinical endocrinologist who specializes in Obesity and type 2 diabetes (Figure 4). Dr. Zhang pointed out that the current AOMs she usually uses in her medical practice, such as Orlistat and Semaglutide, could generally generate approximately 5-15 kg loss of body weight, which is still far from the actual needs of the patients, especially those with a BMI over 30. These weight losses are also accompanied by a set of side effects such as Oily rectal discharge, Vitamin D deficiency, Fatigue, and Nausea, which severely affect the daily life of the patients. More importantly, she pointed out that current AOMs are generally redirected to other drugs, such as anti-obesity drugs and anti-depression drugs, which is probably one of the most important reasons for the high occurrence of the side effects. Through our discussion, we thought that it would be beneficial to design an AOM that specifically targets adipose tissue, preferably using some inherited mechanisms in the adipocytes. This discussion directly inspired us to choose targeted delivery of UCP1 into adipose tissue as the major approach of our therapeutic design (See our Description and Design Page for more information).

With the targeted delivery of UCP1 as our general specification, we then start decomposing this design by deciding the proper delivery approach. Apparently, adipose-specific UCP1 activation can be achieved via either AAV-based gene delivery or by some cell-based approach that secret paracrine factors such as Fgf21 that activate UCP1 expression in the target cells. By approaching multiple scientists who specialize in gene therapy and cell-based therapy, we obtained some inputs regarding the pros and cons of these delivery approaches. Though AAV-based gene delivery could result in robust activation of given genes in the target tissue, the finetuning of AAV-mediated gene expression level remains difficult. Also, AAV injection results in the long-term existence of ectopic genes in human cells (Venditti, 2021), which may raise safety and ethical concerns. In our specific case of UCP1, the constitutively high UCP1 expression level generated by AAV-mediated gene delivery could result in strong toxicity to the host cells. As for the cell-based therapy, according to the experience of Mr. Deyang Zhou, a postgraduate student focusing on mammalian cell Synthetic biology, could encounter more safety and ethical problems as the most widely used chassis, HEK-293T cells, are considered immunogenetic in most cases.

In our additional interview with Dr. Jingjing Zhang, we mentioned our major concern regarding current approaches and asked her about her opinion, as a clinical physician, on gene-based or cell-based approaches to treat obesity. She raised an important point that though these techniques looked interesting, patients are unlikely to accept a gene- or cell-based therapy simply to deal with obesity. She mentioned that the protein- or small-molecule-based approach might be preferable for both patients and administrations.

These findings directly pointed us to finding some protein-based approaches to deliver UCP1 into adipose tissues. Fortunately, we found a timely publication by Kreitz et al. in April 2023, in which they described PVC, a syringe-like protein complex that can effectively deliver UCP1 into the target cell. Hence, we eventually decided to use PVCs as our major delivery approach for UCP1 (See our Description and Design Page for more information).

Engineering a newly reported system is never easy (See the Engineering and Results Page for our achievements!). Multiple technical difficulties were encountered when we were trying to engineer the UCP1-delivering PVC system. On the one hand, the pPVC plasmid carrying the structural and accessory protein is way too large (>25 kb) to be handled using classical cloning techniques since the gel extraction and PCR turn out to be extremely difficult with these large fragments. In our discussion with Mr. Deyang Zhou, he mentioned that we could possibly try to generate a Golden Gate plasmid, in which two Type IIS sites are added into the tail fiber protein, so one preparation of the plasmid backbone would allow us to clone multiple constructs (See our Engineering Page for this design). He also believes that this way can contribute a tool plasmid to the society so everyone in the Synbio community that need to engineer the tail fiber of PVC can possibly use.

Similar points were also made by Drs. Lingbo Li and Mingxing Zhou, two synthetic biologist who works closely with pharmaceutical companies (Figure 6). They mentioned that the current strategy we are using to purify the PVCs is too complicated to be efficiently used in a factory. They suggested that we try to engineer the other structural proteins so affinity magnetic beads can be applied to efficiently collect the PVCs.

To think about the future implementations of our project, we approached Dr. Yaxi Zhang, an expert in drug testing, Dr. Jing Liang, the vice-general manager of a pharmaceutical company, and Mr. Xuyong Xiao, the Chairman of the pharmaceutical company (Figure 7). From the industrial aspect, they suggested us to develop specialized biochemical assay to help controlling the quality of the purified PVCs. They also provided us with key suggestions regarding the animal experiments we might need before we push the project forward to clinical trials. We have delineated a complete development process cycle for ARROW. Please view the Implementation page for more details.