According to ICD-11$^1$, ASD is characterized by persistent deficits in the ability to initiate and sustain reciprocal social interaction and social communication and by a range of restricted, repetitive, and inflexible patterns of behavior, interests, or activities that are clearly atypical or excessive for the individual's age and sociocultural context. In order to help people better understand the symptom of ASD, we referred to literature and visited education institutions for children with ASD, and made a picture book about the neural development disorder. The picture book is available on our homepage.

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The prevalence of ASD has been increasing during the past few years. By 2018, 1 in 44 children had been identified with ASD, according to estimates from American CDC's Autism and Developmental Disabilities Monitoring (ADDM) Network$^2$. In China, the most extensive population-based study to date reports an estimated 0.70% prevalence of ASD among 6- to 12-year-old children. This estimate translates into a total of approximately 700,000 children aged 6–12 years with ASD in China, based on the national census data for 2016$^3$.

the prevalence of ASD according to ADDM

Every child or adult with autism has unique strengths and faces different challenges, so there is no one-size-fits-all approach to autism treatment and intervention. According to American CDC, current treatments approaches for autism include: behavioral approaches, developmental approaches, educational approaches, social-relational approaches, pharmacological approaches, psychological approaches, and complementary and alternative approaches$^4$.

As to pharmacological approaches, no existing medications treat the core symptoms of ASD. Some medicines treat co-occurring symptoms that can improve people with ASD clinical symptom. For example, medication might help manage high energy levels, inability to focus, or self-harming behavior, such as head banging or hand biting. Medication can also help manage co-occurring psychological conditions, such as anxiety or depression, in addition to medical conditions, such as seizures, sleep problems, or stomach or other gastrointestinal problems.

In our first year with the iGEM competition, NJMU-China 2020, proposed a domestic autism screening method based on urinary metabolites in view of the problems of screening in autism. The project “Sound of Silence: Domestic autism screening based on urinary metabolites” stood out among 256 teams and won the Gold Award.

pic:iGEM NJMU-China 2020 project

The following year, in 2021, NJMU-China 2021, their attention from the diagnostic track to the therapeutic track and really got into the depth of autism. They designed engineering probiotics that can both prevent autism caused by maternal infection during pregnancy and treat children with autism. The project “Stairway to starlight: Maternal prevention and offspring treatment of autism induced by maternal infection during pregnancy” stood out in 350 teams, won the Gold Award, and was nominated for the best inclusivity award.

pic:iGEM NJMU-China 2021 project

The previous two NJMU-China teams had interviewed many parents with a child with ASD as their members kept exploring the therapeutic and diagnostic approaches to the neural development disorder. This year, we got our inspiration from revisits to the parents of autistic children that we had been following throughout the years. We learned from our conversations that their expenditures on supplements were relatively high. Even worse, some families were having a more challenging time during lockdown due to the Covid-19 pandemic. During our voluntary work at Star of the Sea, a rehabilitation center for autistic children, we consulted educators and caregivers and were surprised again at how common supplements are applied among autistic children. This led us to form our original idea of engineering our bacteria to produce elements of the most frequently used supplements for autistic children.

revisits to the parents of autistic children

In China, children with ASD are often called “children from the star.” With the experience from the past two years and our human practices this year, team NJMU-China 2022 chose to continue working on the treatment of ASD using synthetic biology approaches and doing our part in magnifying the light from the stars of autistic children.

To date, the etiology of ASD remains unknown. Yet, according to studies, it possibly involves a wide range of environmental factors that affect many physiological processes in genetically susceptible individuals.

Recently, lines of evidence have shown possible contributions of the intestinal microbiota to ASD pathogenesis. With the investigation of the microbiota-gut-brain axis, treating neurological disorders such as ASD through microbiota-mediated metabolites now seems to be a promising approach. Meanwhile, growing evidence has suggested the importance of mitochondrial dysfunction in the pathogenesis of ASD. A controlled study showed that mitochondrial dysfunction might be present in up to 80% of children with ASD$^5$. Toxicant exposure has also been epidemiologically confirmed as an important etiological factor of ASD$^6$, and people with the disorder often show some clinical manifestations of intoxication.

However, how these factors interact with each other and affect ASD pathogenesis is not fully understood.

During our brainstorming, a study drew our attention. Using a quasi-paired cohort strategy, it reveals the impaired detoxifying function of microbes in the gut of autistic children. It suggested that various genetic and environmental factors, such as altered diet and defects in the digestive system, can cause microbial detoxification deficiency, and more toxicants may enter the circulation. The accumulation of toxicants in tissues can potentially cause injury in mitochondria, compromise their function, or harm the integrity of other structures, representing the featured manifestations of ASD$^7$.

pic: Role of impaired microbial detoxification in the pathogenesis of ASD

Pathway Enrichment Analysis

To further study the correlation between autism and energy pathways, we combed global public databases for pertinent information. Ultimately, 1,611 genes associated with autism were discovered in the SFARI database. We used David Database to do the pathway enrichment analysis. The Go functional enrichment analysis identified 342 biological processes (BP), of which 389 genes in 10 pathways were connected to energy metabolism. We drew the following difference analysis diagram using the ggplot2 program in R.

pic: difference analysis diagram

This diagram explains in detail the intimate connection between autism and energy metabolism, which serves as the basis for our project.

Combining the background information and our pathway enrichment analysis, we wanted to establish a novel and promising treatment for ASD that target mitochondrial dysfunction.

As to project design, we aim to improve mitochondrial function and prevent the possible accumulation of heavy metals. Moreover, we intend to build a drug delivery system for our engineering bacteria and create a testing strip to diagnose mitochondrial dysfunction in autistic children. To reach the above four goals, we designed four systems to solve the problems correspondingly and conducted experiments to verify their function.

Wet Lab

The purpose of the wet laboratory experiment is to materialize our concepts. We begin by selecting the suitable chassis, finding the relevant sequences, and synthesizing them. Then, we transformed the synthesized plasmid into chassis bacteria, the success of which was proved by agarose gel electrophoresis. For each system, we designed a variety of experiments, including WB, Elisa, and growth curves to verify the function of bacteria. Simultaneously, we registered the part in the iGEM part registry and measured them quantitatively. In addition, we designed suicide switches and tested them for biosafety purposes.

pic: wet lab work conclusion

Dry Lab

Model, software, and hardware are the three sections of our dry lab. In the modeling section, we developed 3D structure analysis, molecular docking, and ODE models to help the wet lab. In the software section, we developed a game that embodies our design philosophy to help the public understand the mechanism of our project. In the hardware section we designed a lactate test kit, which serve our start-up and reflect our thinking about the proposed implementation.

pic: dry lab work conclusion

Human practices

In order to build a project that is good and responsible for the world, we carried out our human practices through four main aspects: pushing our project forward, helping children with ASD, supporting parents of autistic children, and promoting synthetic biology. We considered contacting specialists and stakeholders an excellent way of testing whether we are going in the right direction. Several contacts, including researchers, educators, doctors, entrepreneurs, parents, and autistic people themselves, helped modify our project at different stages. We were able to identify the real problem and test the feasibility of our project thanks to their assistance. In terms of education, we managed to reach people of various social and educational backgrounds and attached great importance to the accuracy of the information we provided. We also focused on the delivery of our message and made alterations as we moved to a different group of people. Meanwhile, we tried to promote mutual learning experiences in all our educational activities. We have gone through the whole process of entrepreneurship by October 2022 but still plan to continue working on the commercialization of our project after the competition.

pic:HP work conclusion