《1 Engineering research fronts》

1 Engineering research fronts

《1.1 Trends in Top 10 engineering research fronts》

1.1 Trends in Top 10 engineering research fronts

The Top 10 engineering research fronts in the field of medicine and health include basic medicine, clinical medicine, medical informatics and biomedical engineering, and traditional Chinese medicine (Table 1.1.1).

These 10 fronts also involve “molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2”, “aging mechanism and intervention”, “universal CAR-T cell immunotherapy”, “whole-brain connectomics at the mesoscopic scale”, “gut microbiota and metabolic diseases”, “human phenomics”, “mRNA-based therapeutics”, “single- cell multiomic analysis”, “pharmacodynamic material basis of traditional Chinese medicines and their action mechanism”, and “studies of functional genomics of mental disorders” . All core papers on these fronts published between 2015 and 2020 are listed in Table 1.1.2.

(1)   Molecular mechanism of genetic evolution and cross- species transmission with SARS-CoV-2

The novel coronavirus SARS-CoV-2 is currently the 7th known coronavirus that is pathogenic to humans. A large number of other coronaviruses also exist in nature. Bats and rodents are their natural hosts, and the risk of transmission from animals to humans represents a long-standing risk. A lack of clarity regarding the molecular mechanism underlying cross- species transmission of the novel coronavirus in humans, poor knowledge regarding the distribution, evolution, recombination and mutation of coronaviruses derived from different animals in the natural ecological system, and a lack of clarity regarding the key stages in the viral life cycle, including replication, translation, assembly, and release. The structures and functions of the viral genome and the encoded proteins of the novel coronavirus and other emerging highly pathogenic viruses remain to be determined through the use of structural biology, bioinformatics, and molecular biology technologies. Mechanisms by which the immune system is activated after viral infection and the pathogenesis of acute lung injury and multi-organ failure remain to be investigated. Specific diagnostic markers, risk factors for severe disease, and

《Table 1.1.1》

Table 1.1.1 Top 10 engineering research fronts in medicine and health

《Table 1.1.2》

Table 1.1.2 Annual number of core papers published for the Top 10 engineering research fronts in medicine and health

correlates for immune protection remain to be determined. To cope with the threat of the COVID-19 pandemic, we aimed to systematically study the viral proliferation and infection, and mechanism underlying the pathogenesis of COVID-19, to explore the features of transmission, epidemiology, and outbreak to elucidate the origin, evolution, and mutation, and to develop vaccines and medicines to allow for the safe and efficient treatment of this disease to ultimately provide scientific support for the prevention and control of the pandemic.

(2)  Aging mechanism and intervention

The high incidence of aging-related diseases, coupled with an increase in the aging population, is a major global health issue. The functional decline of the body often accompanies aging. Healthy aging is possible if we can dissect the effect of aging on organ homeostasis, develop systematic evaluations of physiological and organ status, and implement early therapeutic interventions for aging-related diseases. The combined efforts of scientists around the world have led to a series of important advances and breakthroughs in the field of aging research. However, serious challenges remain. Apart from novel technologies, new models that reflect human aging are necessary for analyzing and revealing aging mechanisms. The focus of future research includes analyzing the mechanisms of systemic aging; identifying molecular biomarkers and potential targets of organ aging; revealing the role of immune system in organismal aging; decoding the genetic and epigenetic regulatory mechanisms that contribute to aging; developing programming and reprogramming technologies for aged cells; developing new small-molecule drugs, gene interventions, and cell therapy strategies for aging-related diseases; and exploring and developing new aging intervention methods. In addition, aging research is important for establishing comprehensive and systematic aging consortiums and databases; encouraging multidisciplinary and multi-dimensional multi-level research; promoting the translational application of new technologies into dynamic monitoring, early warning, and intervention strategies for aging-related diseases; refining policy support, institutional settings, and research projects; and popularizing the general knowledge in aging research.

(3)  Universal CAR-T cell immunotherapy

In the field of oncology, CAR-T is a cellular immunotherapy that is currently in hotspots, and it has become an important method for the treatment of some refractory/relapsed hematological tumors. It collects lymphocytes from patients and uses gene editing to engineer them before being reinfused. The engineered lymphocytes, namely, CAR-T cell, can recognize tumor antigens independently of major histocompatibility complex (MHC) restriction to kill tumor cells efficiently. However, traditional CAR-T cell also has limitations, for example, CAR-T cell is prepared from patient’s lymphocytes, making product quality difficult to guarantee. Moreover, CAR-T is expensive, and it has a long production cycle. It can also cause antigen escape and toxic effects in clinical application. Unlike traditional CAR-T, universal CAR-T therapy uses T cells from healthy donors. The human leukocyte antigen (HLA) genes and T cell receptor (TCR) genes of these allogeneic cells have been knocked out to avoid immune rejection and graft-versus-host disease. This therapy also involves installing a universal CAR structure to split the traditional CAR structure into two parts: one part is located in T cells, including the intracellular signaling pathway, transmembrane region, and special extracellular structure; the other part is a protein, which has antibodies that can recognize tumor antigens. This protein will also be recognized by the extracellular structure of T cells, which serves as a connection between T cells and tumor cells, namely, targeting modules—various modules targeting different tumor antigens are universal to the extracellular structure of T cells. Universal CAR-T cell immunotherapy breaks through many of the limitations of traditional CAR-T and becomes the forefront of CAR-T cell immunotherapy.

(4)  Whole-brain connectomics at the mesoscopic scale

The brain is an important organ of the human body. Brain science explores the essence and principles of cognition, consciousness, and intelligence. The connectome—a comprehensive map of neural connections in the whole brain—charts the structural and functional connections among single neurons, neuronal populations (i.e., nodes within a functional network), or brain regions, and it is the foundation to comprehensively understand the mechanism of the brain. Thus, assembling brain-wide connectivity maps has become a pressing challenge in brain science.

In recent years, brain research has been gradually progressing from understanding the macro-connections across the entire brain at the level of centimeters to millimeters to clarifying the meso-connections between and within brain regions at the millimeter to micron scale and then to comprehending the micro-connections between individual neurons at the resolutions of micron to nanometer. Developments of technological tools such as magnetic resonance imaging have driven the noninvasive exploration of the macroscopic structure and neuronal activities of the brain. However, these macroscale imaging methods have limited spatial range and resolution, and they cannot fully contribute to the structural and functional connections of the brain neural network. A cell- specific spatial resolution (microns) at the mesoscopic level and a high temporal resolution (milliseconds) are needed to map the electrical activity in neuronal ensembles.

As a new interdisciplinary frontier, brain science research programs have been implemented in different countries, albeit with different points of focus. The United States of America (USA) is more focused on developing new brain research technologies, whereas the European Union concentrates on using a supercomputer technology to simulate brain function. Japan has also launched the Brain/MINDS Project, which studies various brain functions and brain diseases using the common marmoset as an animal model.

In 2018, China established a comprehensive program for the China Brain Project, the scheme of which is centered on basic research and on neural circuit mechanisms underlying cognition. China has world-class facilities that operate cutting-edge imaging technologies such as micro-optical sectioning tomography (MOST), which enable the stable large-scale mapping of whole mouse brain connectome at a single neuron-level resolution. This imaging procedure forms the basis for mapping the whole-brain mesoscale connectome and places China at the forefront of the field. Chinese scientists have initiated the big science project “Whole-brain connectomics at the mesoscopic scale,” which proposes to map neuron type-specific whole-brain connectomes at a single-cell level resolution utilizing animal models such as mice and nonhuman primates that are close to humans. This big science project is at the frontier of the scientific exploration, which will provide comprehensive understanding of the role of neural circuit activity in higher cognitive functions. Moreover, this will project the sites for precise diagnostic and therapeutic targeting of neural circuit dynamics in severe brain diseases and provide a framework for innovations and a basis for simulations in the development of artificial intelligence (AI)-related technologies, including brain-like computing models and brain-machine interfaces.

(5)  Gut microbiota and metabolic diseases

Metabolic diseases, such as obesity, type 2 diabetes mellitus, atherosclerosis, hypertension, and cardio-metabolic disease, are common chronic metabolic disorders with immense differences in etiologies and pathologies. In recent years, data from microbiota studies have shown that the risk factors of metabolic diseases may originate in the gut, and such factors are directly related to disease-specific abnormalities in the composition and function of the gut microbiota. The gut microbiota plays important roles in the training of host immunity, regulating gut endocrine function and neurological signaling, digesting food, modifying drug action and metabolism, eliminating toxins, and so on. The bioactive metabolites produced by the gut microbes can enter the bloodstream of the host through absorption by means of enterohepatic circulation, thereby affecting the various physiological functions of the host. Therefore, gut microbiota dysbiosis has been implicated in the etiopathogenesis of multiple diseases. Moreover, as a potential therapeutic target, targeted interventions on gut microbiota have led to disease prevention and treatment. The etiology of metabolic disease-associated aberrant gut microbiota, the in-depth understanding of the influence of gut microbiota on host metabolism, the mechanism of mutual regulation between gut microbiota and host during disease progression, the specific gut microbes and microbial derivatives that can be used as biomarkers and therapeutic targets for disease diagnosis and treatment, and the disease-specific and patient- specific precise interventions are key scientific issues that need to be solved. Although gut microbiota is considered as a central driver of dysmetabolism, no “gold standard” reference of a human gut microbiota can promote host’s health or disease. This finding is due to a tremendous variation among individuals of different demography, diet, lifestyle, sex, age, etc. In addition, the composition and function of the gut microbiome at various anatomic sites are different and complex. Therefore, gut-centric metabolic disease research aims to clarify the variation rules of gut microbiota in different phenotypic metabolic diseases and then reveal the etiopathological significance mechanism of those common and disease-specific functional changes to the host. These results can also open the possibility for microbial pathways as mediators and potential pharmacological targets for the treatment of metabolic diseases. Thus, clinical research on precise and targeted modulation of gut microbiota through prebiotics, probiotics, targeted antibacterial agents, etc. will promote the prevention and treatment of metabolic diseases. At present, research on gut microbiota in different metabolic diseases has been conducted in China. However, most of the cross-sectional studies only reveal the changes in the gut microbiota of patients, and such studies fail to fully explore the physiological and pathological functions and mechanisms of these changes. In addition, the causal relationship and interaction mechanism between the aberrant gut microbiota and metabolic diseases remain unclear; no targeted microbial drug has entered clinical applications.

(6)  Human phenomics

Phenotypic variation is produced through a complex web of interactions between genotype and environment. Phenome is defined as the complete phenotypic representation of the species, which is the collection of all biological, physical, and chemical characteristics of the human body from embryonic development to birth, growth, aging, and death, including the morphological characteristics, functions, behavior, and rule of molecular composition. Human phenomics aims to precisely measure the human phenotypes and comprehensively analyze the human phenome, which will systematically deconstruct the strong relationship among phenotypes, construct a phenotype network, open up multi-dimensional and cross- scale correlations between the macro and micro phenotypes, and clarify the cross-scale correlation among phenotypes. These factors are important to decode the life sciences, which will facilitate precise health management.

The scientific issues that must be resolved are as follows: ① the development of multi-dimensional, cross-scale correlations and high-resolution analysis technology of human phenome, the establishment of joint application technology system of multi-dimensional human phenotypes, and the construction of relevant national and international standard systems; ② the development of key technologies for cross- scale phenome data analysis and construction of cloud-based one-stop multi-dimensional system for transmission, storage, calculation, analysis, and modeling of phenomics data; and ③ the research to establish phenome analysis technology for critical diseases such as malignant tumors, cardiovascular diseases, cerebrovascular diseases, and metabolic diseases, draw reference atlas of phenome for critical diseases with cross-temporal and spatial resolution, find novel intervention markers of critical diseases, establish risk prediction models of major diseases, and reveal the occurrence and development mechanism of major diseases.

Phenomics can systematically sort out disease information, which may provide medical and health big data. The USA, the UK, and Germany have accelerated their scientific research support for human phenome research, with the constant increase of the number of related research projects in recent years. China has taken the lead in launching the human phenome research project and has systematically deployed its research. The world’s first cross-scale and multi-dimensional phenotyping platform for deep phenotyping and data analysis has been established in China, covering 20 000 phenotypes in 15 categories and providing a one-stop solution to the measurement of human phenotypes at macro and micro levels. At the board meeting of the International Human Phenome Consortium, the participating scientists reached an important consensus that the human phenome big science project should focus on three near-term priorities, namely, phenomics research of new coronary pneumonia and other major diseases, technology system and research infrastructure for phenomics, and standard operating procedures in phenomics research.

(7)  mRNA-based therapeutics

In vitro transcribed (IVT) messenger ribonucleic acid (mRNA) is engineered to structurally resemble naturally occurring mature and processed eukaryotic mRNA. Such synthetic mRNAs deliver genetic information that allows the translational machinery of the host cells to produce many copies of the encoded proteins, which can serve as antigens to boost immune responses or as supplementary proteins, resulting in a therapeutic effect. With recent developments in mRNA in vivo delivery platforms and the improvement of modified regulatory systems, the stability and transfection efficiency of mRNA have been greatly improved. At present, modified regulatory systems utilize caps, 5′ and 3′ untranslated regions, open reading frames, poly(A) tails, and chemically modified nucleotides. The striking effectiveness of mRNA vaccines against COVID-19 not only offers hope to end the current pandemic, but also presents mRNA-based therapeutics as a potential treatment class that may be used to fight against other diseases. To date, mRNA-based therapeutics, including vaccines and protein replacement therapy, are being clinically developed for infectious diseases, cancer, and rare genetic diseases.

mRNA therapy has fast production and low cost, and it is considered safe. However, some basic scientific questions and technical challenges are identified, which need to be addressed for the successful clinical application of mRNA- based therapy. For example, basic principles must be established to optimize the design of mRNA drugs on the basis of the key parameters such as translational efficiency, half- life, and immunogenicity and improve the mRNA stability and delivery systems for targeting specific tissues. With regard to stability, quality control should be improved, and the negative impact of immunogenicity should be reduced. Quality control refers to improving the ability to detect residual template deoxyribonucleic acid, IVT reaction by-products, and incomplete mRNA. We believe that significant advances in basic mRNA biology and delivery platforms will ensure that IVT mRNA can potentially revolutionize the field of vaccine development and the treatment of cancer and rare diseases.

(8)  Single-cell multiomic analysis

Single-cell multiomic analysis refers to the experimental and computational tools analyzing the quantitative changes of various molecular species in individual cells. It is a powerful approach to generate a global view of the cellular activities in normal development and diseases such as cancer. The field of single-cell multiomics is relatively young. Its conception can be traced to the start of the Human Cell Atlas initiative in 2016, which aimed to characterize all cell types in the human being. The advent of single-cell multiomic analysis is facilitated by advances in two technological areas: the maturation of many high-throughput single-cell techniques that can simultaneously survey the genome, transcriptome, proteome, epigenome, metabolome, lipidome, and/or microbiome of thousands-to-millions of cells; the use of new algorithms and bioinformatic tools to integrate large and complex multimodal data to provide new insights into the biological processes that cannot be inferred with a single mode of assay. With these exciting progresses, single-cell multiomics improves understanding of three frontiers in biomedical sciences. First, integrated single-cell data of the genome, transcriptome, and proteome are used to reveal the cis- and trans-regulatory factors maintaining cellular homeostasis and differentiation. Genome-wide association studies (GWAS) are important in identifying causal variants and genes of loci underlying complex genetic traits or diseases. Second, spatial transcriptomics incorporates the positional information of cells in their natural environment with the measurement of gene expression levels in the cell; thus, coordinated movement and interaction among cells could be studied to reveal high-level control mechanisms governing organogenesis. Spatial single-cell analysis is the key to understanding the complicated interplay between tumor cells and the components of the tumor immune microenvironment, which is essential to improve the efficacy of cancer immunotherapy. Third, an increasing number of studies combining single-cell molecular measurements with gain- and loss-of-function methods is found, such as those based on CRISPR/Cas9 gene-editing technologies. Such studies are forming the basis of single-cell functional genomics, with the potential to unravel nuanced, yet critical, insights into the gene control mechanisms of cellular states in normal and abnormal conditions. This approach will lead to the identification of novel targets for therapeutic intervention. To date, the main challenges of single-cell multiomic studies include the effective reduction of experimental noises and dropout rates in data collection, two common technical caveats of most single-cell technologies, and accurate alignment and integration of data derived from different experimental platforms. Moreover, inference of the dynamics of most biological processes requires longitudinal analysis of samples collected at different time points, which necessitates the production of high volumes of data and thus places high demand for quick and efficient storage and analysis of such data. Given the interdisciplinary nature of single- cell multiomics, more cooperation between computational and experimental scientists is necessary. In recent years, many practitioners of single-cell multiomics in China have played instrumental roles in developing sequencing-based single-cell technologies, particularly in sequencing single- cell transcriptomes and genomes. Nevertheless, commonly used experimental platforms of single-cell analysis are still imported from overseas. Many academic groups and commercial companies have been adapting modern single-cell multiomic analysis in studying the life cycle of development and diseases and in screening new therapeutics. Therefore, this burgeoning field will play an increasing role in basic and translational sciences.

(9)   Pharmacodynamic material basis and mechanism of traditional Chinese medicines

The pharmacodynamic material basis of traditional Chinese medicine refers to the sum of the effective components of traditional Chinese medicine, which produce therapeutic effects on a disease, that is, the chemical components (groups) of traditional Chinese medicine, which affect multiple targets and produce overall effects after entering the human body. Its possible sources include the intrinsic components of herbs, products formed during preparation such as decoction, and metabolites produced by the interaction of the medicine with the human body. The effective substances of traditional Chinese medicine could be small molecules, macromolecules, or a combination of both. The effects of traditional Chinese medicine are produced by the combined effects of various effective components (groups) and a variety of disease-related targets. Therefore, the elucidation of the pharmacodynamic material basis of traditional Chinese medicine and its regulatory mechanisms and networks on the targets and the body, as well as the metabolic control of the body on the effective substances, is the key to the interpretation of the overall efficacy of traditional Chinese medicine and its scientific nature.

Traditional Chinese medicine has the advantages and characteristics of multi-component, multi-target, and integrated regulation, and their clinical efficacy is the result of the synergistic effect of multiple active ingredients. Investigation on the pharmacodynamic material basis and mechanism of traditional Chinese medicine should consider not only the multiple effects of traditional Chinese medicine, but also the holistic view of traditional Chinese medicine. However, the following key scientific issues are identified: exploring a suitable model for the study of the pharmacodynamic material basis of traditional Chinese medicine and elucidating the scientific nature of the pharmacodynamic material basis of the traditional Chinese medicine and their medicinal effects. In addition, such scientific issues include the establishment of a comprehensive efficacy evaluation system based on the efficacy of traditional Chinese medicine; the systematic interpretation and precise characterization of chemical components; the determination of the types, structures, contents, and ratios of the bioactive ingredients corresponding to the efficacy of traditional Chinese medicine; and the study of the mechanisms of interaction between the bioactive ingredients and the organism. Therefore, a bioassay system must be established for the overall effects of traditional Chinese medicine, which is a holistic approach to the study of the interaction between active substances and the organism, and the interconnection between “disease-gene-target medicine.”

Study on the pharmacodynamic material basis and mechanism of traditional Chinese medicine is an important part of the modernization of traditional Chinese medicine and a bottleneck for the development of traditional Chinese medicine. The traditional research approach includes the separation of the chemical components guided by bioassay, but multiple components work in synergy to exert the medicinal effects of traditional Chinese medicine. A single component or several components cannot fully represent the efficacy of the entire traditional Chinese medicine. Another approach is conducting activity-oriented chemical composition studies, but they do not reflect the in vivo biotransformation of components, the interactions of medicine with regard to absorption and distribution, and the overall effective substances of traditional Chinese medicine. With the development of a holistic view of traditional Chinese medicine and modern biotechnology, research models led by serum pharmacochemistry, spectrum-effect relationship studies, metabolomics, pharmacokinetic–pharmacodynamic models, molecular biochromatographic technology, network pharmacology, and chemical biology have emerged. In addition, these models have been applied to the study of the pharmacodynamic material basis and mechanism of action, which has promoted the development of traditional Chinese medicine to a certain extent. In the future, advanced technologies and concepts from multiple disciplines must be combined to establish a new paradigm for the study of pharmacodynamic material basis and mechanism of traditional Chinese medicine; comprehensively explain the effective components of traditional Chinese medicine and its chemical communication with living organisms, regulatory network, and scientific nature; and to lay the theoretical foundation for the modernization, international promotion, secondary development, and clinical application of traditional Chinese medicine.

(10) Studies of functional genomics of mental disorders

Mental disorders, accounting for 15% of the total global burden caused by various diseases, have complex symptoms and etiology. Clinically, mental disorders are categorized into organic and non-organic psychosis and manifested in different degrees of mental impairment, including cognition, emotion, motivation, and behavior. Mental disorders are caused by the interaction of genetic and social environmental factors during the development of the body. Functional genomics refers to the study of the dynamic rules of gene encoding and state information transmission and effects, including gene mutation/modification, gene transcription and translation, gene expression regulation, and protein interaction network.

Genome-wide association study of psychiatric disorders is a primary method of identifying disorder-associated genetic factors. The reported disorder-associated DNA variants are often rare and located within non-coding regions of genes, and the findings for a single psychiatric disorder from different studies, such as schizophrenia, are inconsistent. This finding suggests that psychiatric disorders are highly heterogeneous, and new ideas and approaches are necessary to identify disease-causing genes and clarify the mechanisms of function of disorder-associated mutants. Large-scale human samples may subside the impact of individual heterogeneity. However, at present, the number of high-quality bio-banks containing comprehensive clinical and genomic information is limited. Patients with mental disorders are considered as vulnerable groups, and research on their tissue samples requires strict ethical review of scientific research. The animal mental disorder models are recognized as essential tools for studying gene function and mechanism, but only few of these animal models are considered suitable. These two aspects limit mental disorder research. Based on existing human psychiatric data, mathematical models are established to reveal the high sensitivity and high specificity of multigene risk or gene interaction network module of a single psychiatric disease and the relationship among different mental and physical diseases through analyzing the common genetic basis and specific gene function. The effect of single-gene variation in psychiatric disorders is limited, but it may influence a certain endophenotype causing a disease, for example, the substance density of different brain regions and microglial activity. Based on the theory of brain-derived mental disorders, constructing various genetic maps of gene expression and methylation in different brain cells obtained from cadaveric brain tissue samples is essential. On the basis of these maps, cutting edge studies are conducted to identify a gene co- expression network module, clarify the effects of different molecular network modules, such as neural development and immune response molecular networks, on mental disorders, and elucidate the effects of genes in different brain regions and cells at different developmental stages of the human brain and the regulatory function of epigenetic factors, such as methylation, miRNA, and LncRNA. Furthermore, identifying the DNA variation in loci that regulate the molecular quantitative phenotypes, such as expression quantitative trait loci and methylated quantitative trait loci, and analyzing their co-location and disease-associated genetic loci from GWAS are essential to clarify the role and mechanism of DNA variants in the occurrence and development of disorders.

Many scientific research departments in China have established some biobanks and databases related to mental disorders. Some of these departments may not only collect blood samples, but also cadaver brain tissue samples, and some of them work with internationally renowned UK biobanks and have participated in the international PsychENCODE Alliance for data sharing, who aims to conduct large case-control studies with more than 100 000 cases. Some departments have tested and verified the effect and mechanism of gene variation, such as DISC1, on the proliferation of in vitro cultured neurons or microglia derived from patients with psychosis. Some departments have developed new analytical methods to identify and validate the disease-causing genes and molecular interaction networks through integrating different types of genetic data.

《1.2 Interpretations for three key engineering research fronts》

1.2 Interpretations for three key engineering research fronts

1.2.1 Molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2

A global pandemic caused by the novel coronavirus SARS- CoV-2 that humans have rarely encountered in a century. Its genetic evolution and other pathogenic characteristics have reshaped the scientific community’s understanding of the virus. SARS-CoV-2 is undergoing genetic evolution all the time, like other viruses or living organisms. The genetic evolution of SARS-CoV-2 is closely related to the cross-species infection, host adaptation, antigenic variation, pathogenicity and transmission. Till now, far more than 10 species of animals such as bats, pangolins, mink, white-tailed deer, and cats have infected or carried SARS-CoV-2 and SARS-related coronavirus. In particular, the bat coronavirus RaTG13 and BANAL-236, share the highest genome wide sequence identity (over 96%) with SARS-CoV-2 of all known coronaviruses. These researches have revealed the characteristics and mechanisms of cross- species infection, and provided evidence of natural origin theory. In addition, cross-species infection correlates tightly to the pandemic prevention and control strategies in the population for SARS-CoV-2 circulating within different hosts in nature. Moreover, the genetic evolution of the virus itself has played an important role in the cross-species infection，which holds the largest known genome as a RNA virus. Therefore, it is of great significance to understand the cross-species transmission, pathogenicity and other genetic evolution trajectories points, risk points, and host adaptation points of SARS-CoV-2. These findings are highly instructive and crucial for predicting, early warning, preventing and controlling the current COVID-19 pandemic and the next possible infectious disease.

Chinese scientists are taking the lead to carry out researches on the genetic evolution of SARS-CoV-2 and its related viruses, the comparative evolution of them about genetic skeletons, host cell receptor-binding domain, splicing sites of the spike protein, etc. These findings can shed light on the characteristics of genetic evolution as well as the natural origin of SARS-CoV-2. Moreover, Genome-wide genetic evolution analysis of SARS-CoV-2 itself can reveal the evolutionary characteristics of cross-species infection, dynamic evolution of interpersonal communication and network transmission characteristics of the virus, reflect the transmission law of the virus, and construct a hierarchical pedigree division system. By associating the key functional sites in the key evolutionary lineages of viruses with pathogenicity and transmission, we can grasp the adaptive evolution law of viruses. For instance, Chinese scientists firstly proposed that SARS-CoV-2 is divided into two major lineages (L and S lineages) based on two tightly linked mutation sites. This lineage designation has been verified by several subsequent studies, such as A/B typing system study. Studies have also found that there is a host- specific or tissue-specific adaptive dynamic single nucleotide variation iSNV (Genetic tuning) in the process of virus infection in different hosts, which is helpful to reveal the adaptive evolution of SARS-CoV-2 and the characteristics of interaction with hosts.

The research on the cross-species infection of SARS-CoV-2 mainly focuses on the investigation of natural infection reservoirs such as wild animals and domestic animals, the laboratory animal infection models, and the prediction of the susceptibility of virus receptors recognizing animal hosts. The newly discovered SARS-related coronavirus from bats and pangolins have the main molecular characteristics similar to SARS-CoV-2, though they own their unique characteristics. The host receptor angiotensin converting enzyme 2 (ACE2) plays a key role in SARS-CoV-2 cross-species infection. Structural biology revealed the molecular interaction mechanism between ACE2 and SARS-CoV-2 spike proteins. The molecular structure of ACE2 and binding affinity to various species such as human, cat, pangolin, and chrysanthemum bat were further studied. It was demonstrated that the affinity of spike proteins binding to corresponding species receptors was highly correlated with host susceptibility. In addition, scientists have identified the function of other key host factors such as TMPRSS2, TMPRSS4, Neuropilin-1, CD147 and GRP78 during SARS-CoV-2 infection. The research on key host factors and their co-evolution with viruses is of great significance for the study of potential natural hosts and intermediate hosts and the evaluation of the role of different species in SARS- CoV-2 cross-species transmission.

In addition, considering the continuous discovery of coronaviruses that can bind to human ACE2 receptors, the wide geographical distribution of rhinolophid bats, and the multi-species susceptibility to SARS-CoV-2, the risk of SARS- CoV-2 in wild animals jumping through the species barrier to infect humans will constantly exist. At the same time, SARS- CoV-2 variants such as Delta have emerged and have new virological characteristics. Cross-species transmission may also play a role in the emergence of mutant strains, which brings new challenges to the effectiveness of vaccines and pandemic prevention and control. Revealing the genetic evolution law of SARS-CoV-2 and grasping the key factors of cross-species infection are conducive to the development of intervention methods for virus evolutionary mutations and cross-species infection.

At present, the crucial scientific problems about the molecular mechanism of the genetic evolution and cross- species infection of SARS-CoV-2 includes: natural hosts and potential intermediate hosts of SARS-CoV-2; key factors and ecological mechanisms of SARS-CoV-2 or related viruses in the initial cross-species infection from animals to humans; genetic evolution and its function during cross- species infection of SARS-CoV-2; other potential threatening coronaviruses in nature; evaluation of cross-species risk from the perspective of genetic evolution; the occurrence regularity of adaptive variation and its accurate prediction on SARS- CoV-2 replication and transmission; the co-evolution law of coronavirus and host; the gene frequency regulation of SARS- CoV-2 and the determination of key host factors in cross- species infection; the development of cross-species infection- related interventions, etc.

In general, the research trend which focused on the division of SARS-CoV-2 variation pedigree, dynamic tracking of global evolution, identification and functional analysis of key adaptive sites, risk assessment and prediction and early warning, have diverged into the identification of adaptive mutation system and its transmission prediction, the gambling between the viral genetic evolution trajectory and the existing interventions, the mechanism of co-evolution between coronavirus and host, as well as cross-species transmission.

Research hotspots include: ① natural origin of SARS-CoV-2 and its ecological mechanism of cross-species transmission; ② risk assessment of SARS-related coronavirus cross-species transmission in wild animals, surveillance of reversed SARS- CoV-2 cross-species transmission and evolution; ③ potential host prediction of SARS-related coronavirus or SARS- CoV-2 variants, prediction and verification of transmission, pathogenicity and immunogenicity after variation; ④ global evolution dynamics and driving factors of SARS- CoV-2 variation; ⑤ prospective prediction of SARS-CoV-2 adaptive mutation selection, including the driving force of immune escape and evolution under different vaccine strategies, and the recommendation strategies of more efficient and broad-spectrum SARS-CoV-2 vaccine strain; ⑥ the impacts and interventions on viral susceptibility, tissue specificity, and pathogenicity and intervention caused by the discrepancy of host factors, such as viral receptors in different species, tissues and time-space; and ⑦ based on the evolution of the virus and key host factors, the development of generic drugs, vaccines, detection methods and non-drug interventions for SARS-CoV-2 variants and viruses capable to cause potential pandemic.

In the research front of “molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2”, the USA and China are clearly in the leading position, the UK and Germany rank third and fourth among the top countries publishing core papers. The citations per paper are distributed between 105.35 and 552.46 (Table 1.2.1), which fully shows that this research front has drawn high attention. In terms of the cooperation network among countries, many countries have carried out comprehensive and extensive collaborative research. A strong correlation was found between the USA and China and the Top 10 countries all have cooperative relations (Figure 1.2.1)

The Top 10 institutions publishing core papers of “molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2” are mainly from China and the USA. The research institutions from China include Chinese Academy of Sciences, the University of Hong Kong, Fudan University, Chinese Center for Disease Control and Prevention,

《Table1.2.1》

Table1.2.1 Countries with the greatest output of core papers on “molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2”

《Figure1.2.1》

Figure1.2.1 Collaboration network among major countries in the engineering research front of “molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2”

and Sun Yat-sen University. The research institutions from the USA include Harvard University, University of Washington, University of Oxford, and University of Pittsburgh (Table 1.2.2). In terms of the cooperation network, it can be seen that collaborative relationship existed among all institutions (Figure 1.2.2).

Based on the above statistical analysis results, as for the research frontier of “molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2”, China currently shows a high quality of academic ability, in parallel with that of foreign countries. And some advice is recommended as follows.

1)   Develop innovative algorithms based on population genetics and system development to continuously track the mutation and global evolution dynamics of the SARS- CoV-2 genome, identify adaptive mutation sites, establish an adaptive mutation evaluation system, analyze the biological effects of mutations in multiple dimensions, systematically construct the genome variation knowledge database, investigate the genome evolution, trajectory of viruses, animals and populations.

2)  Lead the rapid acquisition and sharing of viral sequences, clinical data and samples, promptly detect new important mutation sites and strains and their pandemic trends in populations or animals, and evaluate their pathogenicity, transmission and immunogenicity in laboratories; explore the relationship between viral evolution and immune escape under different vaccine strategies; develop generic drugs,

《Table1.2.2》

Table1.2.2 Institutions with the greatest output of core papers on “molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2”

《Figure1.2.2》

Figure1.2.2 Collaboration network among major institutions in the engineering research front of “molecular mechanism of genetic evolution and cross-species transmission with SARS-CoV-2”

vaccines and detection methods for SARS-CoV-2 variants and other coronavirus that infects humans.

3)  Carry out extensive international cooperation and monitor wildlife animal virus in hotspot countries and regions where wildlife carrying SARS-related coronavirus are distributed; evaluate the possibility of different animal species as potential natural and intermediate hosts of SARS-CoV-2 and further find out the origin of SARS-CoV-2 cross-species spillover; develop new methods to assess the risk of different coronavirus infections in humans and other wild animals, and develop prospective pandemic warning systems, prevention and control technologies, vaccines and drug reserves.

4)    Integrate the advantages of evolutionary biology, virology, bioinformatics, and other disciplines, and combine artificial intelligence and big data analysis to promote the intelligentization of infectious diseases prediction and early warning and the precision of prevention and control strategies, strengthen the research and development of diagnosis and control methods based on key host factors, and summarize the experience and shortcomings of fights against COVID-19, to cope with potential future pandemics.

5)  Make policies to provide long-term and stable support for prevention and control technologies and basic research on pathogens with potential pandemic risks such as coronavirus and influenza virus. Encourage industry-university-research integration in the areas of prospective early warning, non- drug interventions and diagnosis, drug and vaccine research and development, and strengthen policy support.

1.2.2 Aging mechanism and intervention

As of November 1, 2020, the Chinese population aged 65 years or above reached 190 million. This number accounts for 13.5% of the total population, indicating an increasingly old populace. Population aging and the high incidence of aging- associated diseases have been recognized as an emerging issue that is relevant to society and biological research in China and globally. Aging manifests as a functional decline in physiological homeostasis, which increases with age, affects most of the tissues and organs of the body, and augments the risk of a variety of chronic diseases. The systemic aging of tissues and organs throughout the body and the decline in regenerative capacity are thought to be the main reasons leading to aging and the occurrence of age-associated diseases. In-depth and integrated analysis of the impact of aging on organ homeostasis, constructing an index for organ aging, and establishing diagnosis and intervention strategies for aging-related diseases are prerequisites that would promote healthy aging.

Extensive studies based on classical model organisms have provided insights into aging. However, given the species differences, advancing translational research in aging urgently requires the establishment of primate-based research models. Our current understanding of how aging favors the onset of multiple diseases is limited. Thus, further insight into relevant key regulators and molecular targets is necessary. In addition, the interaction between key molecular and physiological factors and age-related disorders remains unclear. These aging-related interactions may have important temporal and spatial relationships and variable outcomes in different individuals. Therefore, the interplay between environmental conditions and genetic backgrounds and the reorganization of crosstalk within networks caused by different signals that contribute to degeneration may selectively make a specific molecule or a cell type critical in regulating aging at specific stages. Under these circumstances, aging regulation may differ case-by-case, thereby increasing the complexity and difficulty of studying aging. Therefore, revealing the molecular mechanisms underlying the development of aging and aging-associated disorders requires focus on senescent or dysfunctional cell types, disturbances in the microenvironment, and various subcellular changes in organelles. These disruptions will lead to disorder of the overall spatial integrative network for aging regulation. The systematic study of different cell types during aging, particularly if such studies are based on non-human primate models and human subjects, would help us explore the fundamental molecular mechanisms underlying cellular aging and their relevance to aging disorders. Such studies could also uncover key regulatory networks and molecular nodes for aging and aging intervention, thereby providing a solid theoretical basis for understanding aging and aging- associated diseases. Moreover, such basic research focusing on aging could provide novel biomarkers and drug targets for aging and aging-related diseases. Therefore, basic research into aging provides an important theoretical and technical foundation for the formulation of effective evaluation and intervention strategies. It will also promote basic and translational medical research for organ degeneration and body aging, thereby leading to the prevention and treatment of aging-related diseases.

In recent decades, with the rapid development of new technologies such as novel stem cell platforms, gene editing, AI for data analysis, and high-throughput methods such as single-cell sequencing and multi-omics, aging research has entered a new era. Such an intervention would delay the aging of organs and the body and prevent aging-related diseases. Current studies have shown that metabolic reprogramming, small-molecule intervention, young blood factors, and gene intervention can delay the aging of cells, organs, and individuals.

Therefore, identifying key factors, regulatory networks, and intervention targets for cellular aging and its reprogramming can provide an important theoretical and technical foundation for the development of effective strategies to delay organ aging and body aging. This research would also promote related basic and translational medical research and achieve effective prevention and treatment strategies for degenerative diseases. Achieving these goals is critical for coping with the current challenges of an aging population, and these goals serve as an important strategic blueprint for the life sciences.

The key questions related to understanding aging mechanism and intervention in aging-related disorders are summarized as follows:

1)  Establishment of novel research models and technologies for studying aging mechanism and intervention. Model organisms play a fundamental and vital role in biomedical studies. However, traditional animal models such as mouse exhibit differences in anatomy and physiology compared with humans. These differences pose a major challenge for translating research findings into clinical treatments for human aging. In addition, aging is a highly heterogeneous and complicated process that occurs over the entire lifespan and involves multiple organs and systems. This intricacy hinders the investigation of the underlying mechanisms of aging. Given these challenges, development of novel methodologies and technologies and interdisciplinary research approaches are necessary for successful aging research.

2)   Systematically decoding novel molecular mechanisms of aging. Previous studies have indicated that aging might be due to multiple factors, including stem cell exhaustion, chronic inflammation elevation, genomic instability, telomere attrition, DNA damage, epigenetic alterations, and metabolic dysfunction. The regulatory networks that connect these factors and key hub factors of aging have not been systematically decoded. Therefore, identifying aging-related biomarkers and reliable and effective targets for intervention is important. Effort should be made to combine the diverse techniques used in biology, medicine, informatics, and AI. Such an interdisciplinary approach is critical to translating fundamental aging research achievements into clinical application.

3)  Development of a new strategy to evaluate and delay aging and aging-related diseases. Novel aging biomarkers would also be conducive to evaluating the progress of aging. Such biomarkers would allow early diagnosis and help evaluate the performance of clinical intervention strategies. Moreover, development of reliable and effective methods targeting aging and aging-related diseases is a major goal of aging research, which should lay the foundation for achieving health aging.

Future research should focus on ① studying the regulatory modality of systematic aging, ② discovering molecular biomarkers and potential targets for organ aging, ③ investigating the effect and molecular mechanism of immune system and organism aging, ④ decoding the genomic and epigenomic mechanisms of aging, ⑤ developing strategies for programing and reprograming of senescent cells, ⑥ developing novel small-molecule drugs and genetic manipulation strategies against aging and aging- related diseases, ⑦ developing (stem) cell therapy for aging and aging-related diseases, and ⑧ establishing novel aging interventions based on active health (e.g., calorie restriction, rhythmic regulation, and beneficial exercise).

Regarding the research front of “aging mechanism and intervention”, the USA has the highest number of core papers being published, whereas China and the UK ranked second and third, respectively, with citations per paper ranging from 61.65 to 146.69 (Table 1.2.3). The citations per paper from China is 80.22. Cooperation exists among the Top 10 countries with the most core papers. These countries participate in a network of collaborations across nations, and the top three countries (the USA, China, and the UK) have strong interactions with one another (Figure 1.2.3).

The Top 10 institutes with the most core papers related to “aging mechanism and intervention” are located in the USA, China, and the UK. Institutes from the USA include the Mayo Clinic, Buck Institute for Research on Aging, Salk Institute for Biological Studies, University of Pennsylvania, and Scripps Research Institute. Institutes from China include Chinese Academy of Sciences and Capital Medical University. Institutes from the UK include Newcastle University, University of Cambridge, and University of Glasgow. Among these institutes, Mayo Clinic and Chinese Academy of Sciences share first place in publishing core research papers (Table 1.2.4). In addition, the institutes involved in the research front of “aging mechanism and intervention” have established cooperation with one another (Figure 1.2.4). The above-mentioned analysis revealed that China is at par with foreign countries in the research front of “aging mechanism and intervention”.

Based on the aforementioned statistical data on leading research on “aging mechanism and intervention”, we propose the following approaches:

1)  Establishing aging-related biobanks and databases with a special focus on non-human primate and clinical samples for aging research. Aging-related biobanks and databases would provide valuable resources for the elucidation of molecular mechanisms and clinical intervention strategies against aging- related diseases and support for increasing the healthspan and life quality of the elderly and promoting sustainable social and economic development.

2)   Encouraging interdisciplinary research collaborations

《Table 1.2.3》

Table 1.2.3 Countries with the greatest output of core papers on “aging mechanism and intervention”

《Figure 1.2.3》

Figure 1.2.3 Collaboration network among major countries in the engineering research front of “aging mechanism and intervention”

《Table 1.2.4》

Table 1.2.4 Institutions with the greatest output of core papers on “aging mechanism and intervention”

《Figure 1.2.4》

Figure 1.2.4 Collaboration network among major institutions in the engineering research front of “aging mechanism and intervention”

involving medicine, biology, chemistry, and bioinformatic approaches to aging mechanism and intervention.

3)  Emphasizing the application of cutting-edge technologies to aging research. These technologies include gene editing, single-cell sequencing, high-resolution imaging, and AI. These approaches can elucidate aging mechanisms, identify new targets, and develop new drugs against aging and related diseases.

4)  Establishing national research institutions focused on aging and aging-related diseases. This step would promote goal- directed programs and provide relatively long-term research support funds from government-funded projects, private capital, and charitable sources.

5)   Strengthening the protection of intellectual property across the life cycle and promoting translational applications via collaborations between researchers and clinicians. We also propose encouraging industrial involvement in scientific assessment, dynamic monitoring, early diagnosis, and therapeutic interventions to help fight aging-related diseases.

6)  Developing silver hair economy by actively promoting the applications of aging research findings. These findings would help the development of new technologies and intelligent products for aging management and facilitate the scientific system construction of the health and well-being industry.

7)  Promoting public understanding of cutting-edge findings in aging research using broadly accessible multimedia approaches.

1.2.3 Universal CAR-T cell immunotherapy

In the field of oncology, immunotherapy has become the fourth type of tumor treatment model after surgery, radiotherapy, and traditional chemotherapy, and cellular immunotherapy has been developed on the basis of this model. CAR-T is the current hotspot of cellular immunotherapy. It collects lymphocytes from patients and uses gene editing to engineer them before being reinfused. The engineered lymphocytes, namely, CAR-T cell, can recognize tumor antigens independently of major histocompatibility complex (MHC) restriction to kill tumor cells efficiently.

CAR-T has significant effects on hematological tumors; thus, it is considered as an important method for the treatment of some refractory/relapsed hematological tumors. However, traditional CAR-T cell is obtained by engineering patient’s own lymphocytes: in the clinic, patients receiving CAR-T cell therapy have undergone multi-line chemotherapy, and the number and quality of cells have declined because of the influence of drugs; patient’s tumor cells secrete a large number of regulatory factors, which will also damage the anti-tumor ability of immune cells in the long run; for elderly patients and those with chronic viral infections, their lymphocytes are characterized by senescence and decreased clonal polymorphism, which are detrimental to CAR-T cell expansion and tumor killing. In addition, tumor cells in lymphocytes are collected from patients. If the CAR structure is integrated into tumor cells, then the target antigen may be abnormal and may not be recognized by CAR-T cells. With regard to production, the individuality of the production is not conducive to the quality control of CAR-T cells; thus, the adverse drug reactions are different, which pose challenges to the safety of clinical applications; the individuality will also hinder the realization of industrialization, making it difficult to achieve high-efficiency, low-cost, and automated production. In the clinical application of traditional CAR-T cells, antigen escape remains a challenge. That is, after the selection from CAR-T cell, the surviving tumor cells no longer express the targeted antigen (or the antigen can no longer be recognized), which will eventually lead to relapse. Although methods such as producing CAR-T cell for other targets or producing bispecific CAR-T cell can cope with antigen escape, they are expensive and inflexible. Toxic effects such as cytokine release syndrome, neurotoxicity, and myelosuppression are also considered as challenges faced by CAR-T in clinical applications, and methods for timely and effectively controlling toxic remain to be explored.

Therefore, the development and application of universal CAR-T cells has become an inevitable trend. “Universal” is embodied in the production of universal T cells and the installation of universal CAR structures. The production of universal T cells refers to collecting T cells from healthy donors, knocking out human leukocyte antigen (HLA) genes and T cell receptor (TCR) genes to avoid host immune rejection of the infused allogeneic CAR-T cells and graft-versus-host disease. Installing a universal CAR structure can split the traditional CAR structure into two parts: one part is located in T cells, including the intracellular signaling pathway, transmembrane region, and special extracellular structure; the other part is a protein, which has antibodies that can recognize tumor antigens. This protein will also be recognized by the extracellular structure of T cells, which serves as a connection between T cells and tumor cells, namely, targeting modules—various targeting modules targeting different tumor antigens are universal to the extracellular structure of T cells.

The application fields of universal CAR-T cell are similar to those of traditional CAR-T, but universal CAR-T has the following advantages. CAR-T cells are produced from engineering healthy human lymphocytes, with stable activity, controllable quality, and high preparation success rate. Its product is more homogeneous, and its adverse reactions are basically similar; thus, CAR-T is safer in practical applications. Consistent production is also conducive to industrialization and automation, which will also reduce production costs and preparation cycles. As a protein, the targeting module is relatively easy to prepare, and it has a low production cost. It can be flexibly replaced during CAR-T treatment to target different tumor antigens, thereby preventing the occurrence of antigen escape. The targeting module can be rapidly degraded in the body. When severe toxic effects occur, the activation of CAR-T cell can be stopped by suspending the input of the targeting module, and the toxic effects can be reduced. These advantages of universal CAR-T have become a research hotspot.

The key scientific issues currently faced by the universal CAR-T are as follows. ① How can universal CAR-T cell, as allogeneic cells, be designed to avoid graft-versus-host disease and rejection better, and how to intervene when such events occur. ② How to optimize the knockout of TCR and HLA genes, which can not only make the knockout more thorough, but also avoid damage to T cells. ③ How to use gene editing to improve universal CAR-T cells. ④ How do universal CAR-T cell differ from traditional CAR-T cell with regard to practical applications, including efficacy, safety, and convenience. ⑤ The screening of lymphocyte donors, that is, whose lymphocytes are used for the preparation of universal CAR-T cell, that is, the screening of lymphocyte donors, and what examinations are required for the cell donors.

The general trend is to promote clinical trials of universal CAR-T cell and increase the number of subjects. The corresponding hotspots are as follows: ① summarize the types and incidence of adverse events in clinical trials, consider short-term and chronic adverse reactions, and explore the best way to deal with adverse reactions; ② improve gene editing by knocking down or overexpression of other genes, inserting foreign genes, and improving tumor killing efficiency to enhance the accuracy and effectiveness of HLA and TCR gene knockout; ③ improve the universal CAR structure, such as adjusting the molecular weight of the targeting module and adding costimulatory ligands to the targeting module to improve its controllability, safety, and effectiveness in clinical use; ④ compare the efficacy of universal CAR-T with traditional CAR-T; and ⑤ explore the application of universal CAR-T cell in solid tumors and other immune diseases.

Among the Top 10 countries producing core papers on “universal CAR-T cell immunotherapy”, the USA ranked first. The UK and France ranked second in the number of core papers, followed by Germany and China. The citations per paper in this research front was 3.22–134.00 (Table 1.2.5), and that of China was 21.00, indicating that the influence of Chinese scholars’ research work in this front still needs to be improved. From the perspective of the cooperation network of countries producing core paper, the top ten countries have cooperative relations within a certain range (Figure 1.2.5).

The Top 10 institutes with the most core papers related to “universal CAR-T cell immunotherapy” include France, the UK, the USA, Germany, and China. The Institutes from France include Cellectis and Servier Research Institute. The institutes from the UK are University College London, King’s College London, and King’s College Hospital NHS Foundation Trust. The institutes from the USA are University of Texas MD Anderson Cancer Center, CRISPR Therapeutics, and University of Pennsylvania. The institute from Germany is GEMoaB GmbH. The institute from China is Henan Cancer Hospital (Table 1.2.6). Based on the cooperation network of the top ten core paper producing institutions, cooperation is observed among the institutions (Figure 1.2.6).

Based on the results of the above-mentioned statistical analysis, China is at par with foreign countries in the research front of “universal CAR-T cell immunotherapy”.

1)   Actively conduct clinical trials, utilize clinical data, and encourage clinical trials under the premise of detailed review and strict implementation of the inclusion and exclusion criteria. By establishing multi-center, transnational platforms or relying on existing platforms, long-term follow-up and

《Table 1.2.5》

Table 1.2.5 Countries with the greatest output of core papers on “universal CAR-T cell immunotherapy”

《Figure 1.2.5》

Figure 1.2.5 Collaboration network among major countries in the engineering research front of “universal CAR-T cell immunotherapy”

《Table 1.2.6》

Table 1.2.6 Institutions with the greatest output of core papers on “universal CAR-T cell immunotherapy”

《Figure 1.2.6》

Figure 1.2.6 Collaboration network among major institutions in the engineering research front of “universal CAR-T cell immunotherapy”

comprehensive data collection could be ensured. After integrating the research data in the platform, the researcher can analyze the safety and effectiveness of the universal CAR-T and suggest practical requirements for its improvement. Detailed research data are also the basis for CAR-T to enter the market.

2)   Prioritize security. CAR-T therapy is quite different from traditional CAR-T. As a cell therapy, it is also different from traditional chemotherapeutics, and it has the uncertainties of biological therapy. Therefore, repeatedly verifying its safety is necessary to protect the interests of subjects.

3)   Learn from traditional CAR-T. The long-term exploration of traditional CAR-T therapy has accumulated considerable results, and its experience in research, development, and application should be learned to improve universal CAR-T therapy. The inspiration of the latter can also be learned by the former.

4)  Encourage multidisciplinary cooperation. Basic disciplines such as immunology and molecular biology should work closely with clinical disciplines. Constantly update gene editing and improve preparation processes in accordance with clinical actual requirements. Cooperation among various clinical disciplines should be strengthened to expand the application of universal CAR-T in solid tumors and other immune diseases.

5)  Promote the modularization of CAR-T therapy. Combining universal T cell with universal CARs, that is, developing a variety of targeting modules to combine with T cells, can make CAR-T therapy more controllable and flexible.

6)  Clarify the principles of donor screening. In the application of universal CAR-T, cells from a certain donor will be allotransplanted to multiple patients, and the condition of the donor will significantly affect the quality of the product. Therefore, reasonable donor screening principles should be determined, and a sound screening process should be established.

7)   Strengthen international cooperation. At present, the academic cooperation in universal CAR-T is primarily between the UK, the USA, and France. Countries should strengthen academic exchanges to promote the development of their own technology.

《2 Engineering development fronts》

2 Engineering development fronts

《2.1 Trends in Top 10 engineering development fronts》

2.1 Trends in Top 10 engineering development fronts

This section of the review describes the Top 10 engineering development fronts in the field of medicine and health, including the fields of basic medicine, clinical medicine, pharmacy, medical informatics and biomedical engineering, public health and preventive medicine, and other subjects. The three emerging fronts are “tumor neoantigen vaccines”, “telerobotic surgery based on 5G networks”, and “RNAi therapeutics”. Traditional research has focused on the development of “clinical translational application of brain– computer interface technology”, “genetically engineered organ xenotransplantation technology”, “combination treatment of targeting multi-immuno-checkpoint inhibitors”, “engineered organoid”, “clinical applications and translation based on the human microbiome”, “health care big data and artificial intelligence”, and “nano-based drug delivery system” (Table 2.1.1). All patents related to these 10 fronts published between 2015 and 2020 have been listed in Table 2.1.2.

(1)   Clinical translational application of brain–computer interface technology

Brain–computer interface (BCI) refers to the direct path between the human brain or animal brain and external equipment, and it can achieve unidirectional/bidirectional information exchange. Brain–computer interface technology is applied to the monitoring, regulation, and function replacement of the nervous system. This technology also aims to control objects and memory storage and transplantation in the future. The key technologies of the BCI can be divided into acquisition, processing, control, and feedback, which have various development speeds. Among them, the control part is relatively mature. The other parts have encountered critical technical problems such as BCI equipment, cognition of EEG signals, neural coding, and functional correspondence. During the development of BCI technology, some branches in clinical translation applications have also been developed, including

《Table 2.1.1》

Table 2.1.1 Top 10 engineering development fronts in medicine and health

《Table 2.1.2》

Table 2.1.2 Annual number of core patents published for the Top 10 engineering development fronts in medicine and health

the detection, diagnosis, and treatment of limb movement disorders, consciousness and cognitive disorders, mental illness, sensory defects, and epilepsy and neurodevelopmental disorders. At present, the domestic and foreign BCI industries are dominated by scientific research institutes, primarily researching non-invasive BCI. The invasive BCI is limited to the medical and health field, and the investment is relatively small. Although the number of companies involved in BCI is less than that of other AI industries, their development trends are still optimistic by companies. In addition, the BCI is still in its infancy at the application landing level. The BCI can gradually establish an open ecosystem in the future. Although BCI companies are rapidly expanding their scale, many companies will also be specializing in the subdivision of the track. In a short time, the medical and health field remains the prominent application market for BCI, and advanced human– computer interaction is difficult to achieve on a large scale in the short term.

(2)    Genetically engineered organ xenotransplantation technology

Organ xenotransplantation is an important way to address donor shortage in the world, and it has experienced many different stages of development. With the prolonged survival of pig xenografts in recent years, its potential clinical application has become increasingly clear. At present, the research and development of genetically engineered organ xenotransplantation still needs to overcome the problems of gene editing site selection, virus elimination, breeding and feeding of donor pigs, immunosuppressant and regimen exploration, evaluation of xenograft function and safety, transplant patient selection, supervision and management, and ethical issues. Several hot sub-fields, including tissue and organ engineering, human–pig chimeric organs, and humanized pig organs, have made important progress and attracted worldwide attention. In the field of humanized pig organs, given the innovation of gene editing and immunosuppressants, relevant pre-clinical research has made breakthroughs continuously, which is widely valued and pursued by the capital. The number of core patents published in the frontier direction of genetically engineered organ xenotransplantation is increasing every year, and it has developed into extensive cooperation among research institutions worldwide. The USA ranks first in patent output, followed by China. Analyzing the current opportunities and challenges faced by our country, we should accelerate the translation of organ xenotransplantation from basic research to clinical application and finally address the critical human health demands.

(3)  Tumor neoantigen vaccines

Vaccines targeting neoantigens represent a novel and potential immunotherapy that induces specific immune responses to eliminate tumor cells. Tumor neoantigens arise from somatic mutations, which are common in tumors, and these neoantigens have strong heterogeneity among individuals. Thus, they can be specific targets for personalized cancer. With the development of bioinformatics and sequencing technology, neoantigens identified by next- generation sequencing have been successfully applied to tumor immunotherapy. A large number of clinical studies have shown that tumor neoantigen vaccine has good application potential in tumor treatment. Vaccines based on neoantigens rather than traditional TAAs have several advantages. First, neoantigens are exclusively expressed by tumors cells; therefore, they can elicit tumor-specific T cell responses, thereby preventing “off-target” damage to nonmalignant tissues. Second, neoantigens are de novo antigens derived from somatic mutations, which present the possibility to circumvent T cell central tolerance of self-antigens and thus induce specific immune responses to tumors. In addition, the specific T cell responses induced by neoantigen vaccines may persist and provide post-treatment immunological memory, thereby increasing the possibility of long-term protection against disease recurrence. In recent clinical trials, neoantigen vaccines significantly prolong the disease-free survival time, bringing new hope to patients with malignant tumors. The diversity of immune responses can be induced by neoantigens, with the numbers and types of neoantigens varying by cancer types, which lead to personalized approach to neoantigen vaccines. The demand for “individualization” of neoantigen vaccines poses new challenges to bioinformatics. The identification of tumor neoantigens and the development of neoantigen vaccines can provide more options for tumor immunotherapy. Neoantigen vaccine combined with other immunotherapy (such as CAR-T or PD1 monoclonal antibody), chemotherapy, or radiotherapy may be an effective way to treat malignant tumors in the future and may play a positive role in promoting contemporary medical innovation.

(4)   Combination treatment of targeting multi-immuno- checkpoint inhibitors

Immune therapy has become the fourth approach after surgery, chemotherapy, and radiotherapy in cancer treatment. Immune checkpoint is an important scientific discovery in the 1990s. CTLA4 is the first immune checkpoint approved by USA Food and Drug Administration (FDA) in 2011. Since then, multiple immune checkpoints have been found. Several antibody drugs targeting PD1/PD-L1 have been approved by the FDA for cancer treatment. In 2018, James Allison and Tasuku Honjo got the Nobel Prize in Physiology or Medicine for discovering CTLA4 and PD1 coinhibitory receptor on T cells. Their important discovery led to the development of immune checkpoint inhibitors. In general, immune checkpoints present a receptor–ligand paradigm. The receptor is expressed on lymphocyte, and the ligand is expressed on immune cells or non-hematopoietic cells, such as APC and cancer cells. PD1 is an important immune checkpoint, and its ligand is PD-L1. PD-L1 binds to PD1 and triggers an inhibitory signal that hinders the cytotoxic effect, allowing tumors to escape from T cell attack. Immune checkpoint inhibitors block the conduction of inhibitory signals by interfering the binding of immune checkpoints to their ligands, thereby enhancing the cytotoxic effect.

Although a considerable fraction of patients demonstrate an objective clinical response, about 80% of patients do not respond appropriately to immune checkpoint inhibitors because of primary or acquired treatment resistance. In fact, tumors can escape from the host immune attack by multiple immune checkpoint pathways, for example, the TIM-3 path, LAG-3 path, VISTA path, TIGIT path, B7-H3 path, IDO1 path, and SIGLEC15 path. Along with more immune checkpoints are characterized, when monotherapy could not get good response, the combination treatment of targeting multi- immuno-checkpoint inhibitors becomes a new option. Recently, the combination of ant-CTLA4 and anti-PD1 in metastatic melanoma has revealed increased overall survival and progression-free survival compared with monotherapy. The responding rate of the combination group is 60%, which is significantly higher than any monotherapy (20%–30%). However, combination regimens are related to higher toxicity. The incidence of immune-related adverse events caused by drug treatment is about 8% for anti-PD1/PD-L1 and 25% for anti-CTLA4, but it increases to 50% in combination regimen of both drugs. Therefore, drug safety is a problem that cannot be ignored.

The FDA-approved immune checkpoint inhibitors are primarily targeted to CTLA4, PD1, and PD-L1. Seven immune checkpoint inhibitors are approved by the FDA: one anti- CTLA4 (Ipilimumab), three anti-PD1 (Cemiplimab, Nivolumab, and Pembrolizumab), and three anti-PD-L1 (Atezolizumab, Avelumab, and Durvalumab). The indications of seven monoclonal antibody drugs covered over 20 kinds of cancers. The combination regimens for CTLA4 and PD1/PD-L1 dual- target have been approved by the FDA for cancer therapy. The alternative combination regimens of anti-PD1/PD-L1 with new generation of immune checkpoint inhibitors are ongoing clinical trials. In China, a total of 17 cancer types are suitable to use immune checkpoint inhibitors according to the guideline by Chinese Society of Clinical Oncology. Eleven immune checkpoint inhibitors are recommended, including four Chinese domestic immune checkpoint inhibitors (Camrelizumab, Toripalimab, Sintilimab, and Islelizumab). From the published data, the overall response rate of monotherapy is not more than 20%. Combination therapy for multiple immune checkpoint inhibitors is a potential strategy, which is in the exploration stage in China or abroad. Accumulating more clinical trials or real-world data is necessary. Meanwhile, exploring the underlying molecular mechanism of multiple targets is necessary to evaluate the risk–benefit ratio of multi-target combination therapy.

(5)  Engineered organoid

Engineered organoids, a combination of bioengineering techniques such as stem cells, biomaterials, and organ-on-a- chip, are used to standardize and automate the production, control, and analysis of human organoid development, homeostasis, and disease modeling by mimicking tissue organoids that perform different functions in vivo, thereby reproducing the complex and dynamic microenvironment of developing organs. Modeling of target organ heterogeneity by organoids is a key technical issue to study. Engineered organoids enable the precise replication of different cell types, structural tissues, and their organ specific biochemical and physiological microenvironments of target organs through bioengineering to model physiological homeostasis at the integrated organ level, complex disease processes, multi- organ interactions, and physiological responses of “body-on- chip” systems, thereby obtaining more complete organoids that resemble in vivo target organs. With the establishment of advanced model systems for bioengineering, engineered organoid technologies provide new functionalities into life medicine research and clinical applications, which show great application potential in various research fields, such as drug research for toxicity detection, pharmacodynamic evaluation, and new drug screening; clinical medicine for establishing disease models to study genetic diseases, infectious diseases, and cancer; and regenerative medicine for studying tissue organ development, transplantation, and repair. Organoids have shown their strong developmental potential globally, and some of the developed countries and regions have promoted the replacement of traditional animal models with in vitro models such as organoids by relevant regulations. A certain market competition pattern has been formed abroad, and the market is expected to reach billions in the next few years. However, the new generation of engineered organ market based on new materials, organ chips, and other technologies is still in its infancy, but the research and development of engineered organs gradually lead to the development of new fields. With the combination of organoids with other sophisticated engineering technologies such as organ chip, micro mobile array, scRNA-seq, CRISPR/ Cas9, high-throughput screening, 3D printing, and intelligent biomaterials, engineered organoids will become more mature with regard to stability, accuracy, reproducibility, and scalability. As a frontier cross-field, engineered organoids have diversified research and development directions and clinical transformation modes. With the continuous maturity of relevant technologies, engineered organoids will excel in translational medicine and clinical personalized treatment and make important progresses in promoting the development of the whole field and overcoming major human diseases.

(6)  Telerobotic surgery based on 5G networks

Telerobotic surgery based on 5G networks is a new telemedicine model that uses 5G communication network as a medical information transmission carrier and a master- slave method to expand the use of robotics, virtual reality, and AI technology and improve remote diagnosis, remote guidance, remote operation, and effect evaluation of medical surgery. This model can quickly and efficiently radiate superior medical resources and improve the efficiency and quality of remote treatment, and it can be used for remote diagnosis and treatment of patients and medical emergency rescue. In performing accurate and safe 5G telerobotic surgery, addressing some key technical issues is necessary, such as real-time feedback and accurate analysis of multi- mode information in the surgical area, human–computer interaction interface suitable for remote operation, and low- latency network transmission. In addition, the standardization of medical information data and security ethical issues must be addressed. With the rapid development of 5G network, the clinical application and promotion of 5G telerobotic surgery are showing a rapid development trend. The scope of clinical application continues to expand, and new clinical procedures continue to emerge, including orthopedics, endoscopic surgery, neurosurgery, cardiovascular surgery and many other fields; the remote surgery model is also innovated, from the “one-to-one” master-slave single-point remote control to networked collaboration modes such as “one-to-many” and “many-to-one”, to achieve not only the simultaneous remote operation of different patients (multiple locations) by one clinical expert, but also the collaboration of clinical experts (multiple locations) in different fields to diagnose and treat the same patient; the application scenarios have also expanded from conventional clinical remote surgery to public health remote diagnosis and treatment services (such as robot-assisted remote ultrasound diagnosis and treatment of patients with COVID-19, remote ward rounds, etc.) and medical emergency rescue (such as natural disasters, space/battlefield, and remote first aid). As the two-core functional hardware of the 5G telerobotic surgery system, medical robotics products and 5G communication networks are maturing and diversified, providing the possibility for a wider range of 5G telerobotic surgery. In addition to some technology start-ups, traditional medical device companies such as Stryker and MicroPort Medical and communications giants such as Huawei and Samsung have also begun to deploy 5G telerobotic surgery products and applications. Related technologies, products, and service markets are also rapidly growing.

The deployment cost of software and hardware for 5G telerobotic surgery is continuing to decrease, providing feasible conditions for the daily development of remote surgery and the expansion of remote surgery applications. Medical AI technology will be further integrated into the 5G telemedicine robot surgery to solve the bottleneck of effective information mining and efficient data transmission of remote diagnosis and treatment big data. Telemedicine ethics and regulatory measures are becoming more standardized, and telemedicine platforms are gradually being systematized and standardized. Telerobotic surgery based on 5G has become an important component of “Surgery 4.0”, which promotes the continuous rapid development of smart surgery, with broad market and application potential.

(7)  Clinical applications and translation based on the human microbiome

Human microbiome refers to the microbial population that inhabits the human body, including its total genes and genomes. It can extend to include the interactions between the microbial population and the host in their niche milieu, which is also collectively referred to as microecology. The technologies for studying the human microbiome include high-throughput nucleic acid sequencing, metabolomics, culturomics, microbiota and host interactions, and bioinformatic analysis. Achieving trans-omics analysis and its verification at different levels on cells, animals, and humans is the key to reveal the structure and function of the human normal microbiome, its interaction networks with the human body, and the underlying mechanism. The relationship among the microbiome, human health, and disease must be further illustrated to maintain health, prevent chronic diseases, and develop new clinical diagnosis and treatment strategies. The human microbiome is closely related to the occurrence and development of chronic diseases and various organ diseases. In addition, it is causally related to reproductive health, infant development, medication effects, and dietary nutrition, providing unprecedented opportunities of disruptive innovation for human health concepts, modern medical theories and diagnosis/treatment techniques, and traditional Chinese medicine theories and practices. The scientific achievements and technologies of the human microbiome have shown broad application potential in the early warning and prediction of various diseases, targeted screening of specific pathogens, precise research and development of targeted drugs, precise nutrition intervention, reproductive health, and traditional Chinese medicine applications. This also provides new perspectives for drug research and development, disease treatment, and prevention strategies and bring the conventional nutrition intervention into a precise individualization stage. Given its broad potential in the military and civilian fields, this emerging field has become a strategic highland for international competition. The USA and the European Union have respectively launched the human microbiome program for nearly 20 years and have achieved fruitful results, spawned many patents, and nurtured several representative companies, thereby developing novel technologies for clinical diagnosis and prediction based on the microbiome, novel therapeutic products of fecal microbiota transplantation and live therapeutics, and novel methods for precise nutritional intervention. Our country has invested in this emerging field for nearly 10 years, but an independent national microbiome research program has not yet been launched. A group of R&D teams have also emerged in the fields of microbiome-based diagnosis, treatment, prediction, and nutritional intervention, and they are among the internationally advanced research fields such as live therapeutics. The market in this emerging field is still in a stage of rapid growth. The precise picture of the structure and function of the human microbiome is a key problem in the development of this field. Multi-omics analysis technology is used to monitor the dynamic changes of the microbiome and its relationship with health and disease and establish a microbiota strain collection bank and a database of the human microbiome and its mining technology. This technology will be the material basis to ensure the clinical translation of this field; interdisciplinary approaches, particularly AI and computer deep learning technology, will promote the genuine integration of complex omics and biological function data and the clinical translation of the microbiome structure and function, and its relationship with health maintenance and disease development will be beneficial to the military and civilian fields in the future.

(8)  Health care big data and artificial intelligence

The integration of health care big data and AI can lead to the exploration of its applications in medical research, clinical diagnosis and treatment, precision medicine, and novel drug research. Thus, health care big data must be collected and collated, including health care service data such as electronic medical records and medical imaging; biomedical data such as genetic sequences and various omics; medical research and development and management data such as drug clinical trials and electronic supervision of vaccination; public health data such as public health prevention and control, and infectious disease reports; statistical data such as health resources, medical service surveys, and family planning statistics. In addition, AI theory and technology, such as machine learning and in-depth learning, can be combined with big data technology, which is a useful and reliable health care research method.

Through a series of technologies, such as statistical analysis, natural language processing, computer vision, machine learning, and bio-information analysis, we can dig for the potential knowledge in the diversified medical big data, such as physiological and biochemical tests, omics data, medical imaging, and electronic medical records. Moreover, we can extract the causal logic among related knowledge, which can be further utilized in explaining, summarizing, and predicting clinical diagnosis and constructing modules within analysis systems and related knowledge. Therefore, our works can be applied in all aspects of medical diagnosis and treatment.

The combination of health care big data and AI technology can be applied to various fields, such as the establishment of a more scientific, automated, and rational diagnosis and treatment and health management system and the construction of a big data platform based on cloud computing and statistical analysis. We have also established a unified multi-functional analysis platform, consisting of an auxiliary diagnosis system based on AI for the identification and discrimination of benign or malignant lesions using medical images, drug discovery and molecular design system, and multi-omics and multi-structured data processing system. According to the McKinsey Rongzhong Research and Consulting Report, in 2020, the Chinese AI market size was estimated at USD 288.4 million pertaining to health care industry, accounting for more than 8% of the overall industry market size. Among them, the application of AI technology in the field of medical imaging is the most extensive and mature. Major hospitals across the country are actively conducting AI research and cooperation projects. In addition, several tertiary hospitals have launched AI technology pilots and clinical trials such as AI image recognition, AI diagnosis and treatment assistants, and AI diagnosis and treatment plans. Based on the data from the consulting firm Frost & Sullivan, China’s AI medical imaging market was estimated at CNY 300 million in 2020, and it is expected to reach CNY 92.3 billion in 2030, at a compound annual growth rate of 76.7%.

With the extensive application of AI technology in the field of health care and the auxiliary support of medical big data platforms, coupled with the increasingly perfect policies and regulations of data application, China’s AI health care industry will also embrace the golden time. Related business will cover data collection, integration, analysis, and application. Thus, data integration-related companies and data application- related companies will achieve explosive growth in the short, medium, and long term. The continuous implementation of big data and AI technology in the medical field will promote the sharing and homogenization of medical resources in China and efficient diagnosis and treatment, thereby providing significant social and economic benefits to our country and achieving the “healthy China” strategy of “co-building and sharing national health.”

(9)  Nano-based drug delivery system

Nano-based drug delivery system is defined as a formulation or a device with size range of tens to hundreds of nanometers, which is applied to solve the bottlenecks of small-molecule drugs, nucleic acid drugs, and protein drugs encounter in in vivo application, including stability, pharmacokinetic properties, and biological safety. Compared with traditional drug administration strategies, the application of the drug delivery system can improve the pharmacokinetic properties of small-molecule drugs, achieve the controlled release of active drug molecules, improve the drug concentration in the lesion site, and reduce systematic toxicity.

Great breakthroughs have been made in basic research in the nano-based drug delivery system, and the clinical transformation is increasing. During clinical transformation, the relatively complex structure of the nano-based drug delivery system requires further evaluation of nano-biological effects, which is different from traditional small-molecule drugs. To date, most nano-based drug delivery systems applied in clinic have relatively simple structures. The lack of systematic investigation of a large-scale production process is the main factor limiting its clinical transformation. Therefore, achieving accurate control of parameters of a drug delivery system such as structure, size, surface charge, targeting ability, and responsiveness through the reasonable molecular design and optimization of the synthetic method and achieving efficient preparation and functional regulation have become a key technical problem restricting the development of a nano-based drug delivery system. In addition, the lack of basic research on nano-biology and the need to improve clinical evaluation methods and related laws and regulations are important factors that limit the development of this field.

In the recent years, with the rapid development of nanotechnology, nano-based drug delivery systems have been applied to the diagnosis and treatment of a variety of diseases, such as cancer, cardiovascular diseases, eye diseases, and neurodegenerative diseases. To date, several nano-based delivery systems have been clinically approved for the treatment of a variety of diseases, showing advantages in therapeutic efficacy, biosafety, and pharmacokinetic properties. At the end of 2019, the outbreak of COVID-19 brings serious threat to people’s health and economic development. Vaccine exploitation is an effective approach to control the epidemic. The coronavirus vaccine based on a lipid-based drug delivery system has been clinically approved, and it has attracted scientists’ attention in academia and business. In addition, RNA nano-vaccine shows good application potential in the anti-tumor area. A nano-based drug delivery system has been considered as the “golden partner” of nucleic acid drugs. Given its broad application potential in the field of medicine and healthcare, China, the USA, and other developed countries in Europe have been improving their investment in this field. The data show that the number of core patents of a nano-based drug delivery system is increasing every year. At present, the number of core patents in China ranks first, but the average citation number of patents is lower than that of the USA, Canada, Japan, and other countries. Six of the Top 10 main institutions that produce core patents are from China, indicating that China pays considerable attention to the development of a nano-based drug delivery system. However, China lacks professional and systematic patent protection strategies. In addition, the USA with many cooperative countries has a relatively high clinical transformation rate, suggesting the need to extend international cooperation.

With the progress of science and technology, the breakthrough of key techniques in production will lead to the achievement of large-scale production of more nano-based drug delivery systems and further promote their clinical transformation. In addition, the extensive cooperation will promote the comprehensive development of materials science, chemistry and chemical engineering, biomedicine, nano science, and other interdisciplinary areas, which will provide new solutions to the key technical problems. The technological breakthrough of a nano-based drug delivery system will certainly bring new options for the diagnosis and treatment of diseases, which can also be applied to several medical fields.

(10) RNAi therapeutics

RNA interference (RNAi) therapeutics are a new drug modality developed on the basis of RNA interference, which is a unique biological phenomenon in eukaryotic cells. Two American scientists who discovered and revealed the phenomenon of RNAi won the 2006 Nobel Prize in Physiology or Medicine. The typical RNAi phenomenon is based on exogenous or endogenous double-stranded RNA oligonucleotide. Single- stranded RNA initially interacts with a specific protein group, namely, RNA-induced silencing complex (RISC) in the cytoplasm, and then it specifically recognizes the targeted mRNA based on the Watson–Crick base complementary pairing mechanism and degrades mRNA to achieve the effect of gene silencing. RNAi therapeutics have a richer and wider selection of drug targets and have characteristics similar to most precise targeted inhibitors. In addition, they show more efficacy and safety than antibody drugs, and they are also conducive to mass production by pharmaceutical companies because of their relatively small molecular weight. In particular, its long-acting effect indicates its potential application in patient medication compliance. Research reports have shown that the R&D for RNAi therapeutics has obvious platform advantages with high productivity. Based on these characteristics, RNAi therapeutics are becoming another new modality of inhibitory drug with great potential after monoclonal antibodies and small-molecule drugs.

The key technologies for the development of RNAi therapeutics are optimizing the active pharmaceutical ingredient (API) and delivering the API safely and efficiently to the target cells of the lesion. The most typical structure of RNAi therapeutics is a double-stranded RNA with a length of 19–25 nucleotides, namely, small interfering RNA (siRNA), in addition to microRNA (miRNA), short hairpin RNA (shRNA), and asymmetric small interfering nucleic acid (asiRNA). All of them can be collectively referred to as “RNAi Trigger”. Chemical modification can enhance the stability of those RNAi triggers in the nuclease-rich environment, optimize drug activity, and reduce their off-target effects. By chemically coupling small-molecule chemical drugs, the modified RNAi drugs can have the dual functions of gene silencing and chemo drug inhibition. The delivery of RNAi therapeutics usually depends on the protection of nanoparticle preparations, such as lipid nanoparticle (LNP), peptide nanoparticle (PNP), polymer polyplex nanoparticle (PPN), and other preparations. Tissue and cell-specific targeting is obtained by conjugating with targeting molecules, such as hepatocyte targeting with an acetylgalactosamine (GalNAc) coupling structure and tumor cell targeting with a RGD coupling structure and specific mAb coupling structure. At present, GalNAc coupling, LNP formulations, and PNP formulations are being used for RNAi therapeutic delivery and tested in multiple clinical trials.

The RNAi therapeutics that have been approved to enter the market are basically limited to liver metabolic diseases. However, a large number of pre-clinical and clinical trial results show that these drugs can be widely used in the treatment of cardiovascular diseases, cancer, ophthalmological diseases, central nervous system diseases, autoimmune diseases, viral infections, fibrotic diseases, and skin diseases. These drugs can also be used in combination with other types of drugs (small-molecule drugs, monoclonal antibodies, and immune checkpoint inhibitors) and treatment options (CAR-T cell therapy, antiviral vaccines, and tumor vaccines) to expand to a wider range of application fields and obtain more efficacious therapeutic benefit.

At present, more than 50 ongoing clinical trials for RNAi therapeutics have been conducted worldwide, which are distributed in different therapeutic areas and at different stages of research and development. Four drugs have been approved by USA FDA onto the market. The main pipeline is currently focused on indications related to liver metabolism diseases, and clinical trials related to oncology are still in the early stages. The global market for RNAi therapeutics will increase from USD 362 million in 2020 to USD 21 billion in 2030. The cumulative investment of large pharmaceutical companies already increased from USD 8.6 billion in 2017 to more than USD 35 billion in 2020. Chinese investment institutions have invested more than CNY 2 billion in RNAi therapeutics development in the past 2 years. In the past 3 years, global capital market returns for investment in RNAi drug have been 400% higher than the S&P 500 Index and the Nasdaq Biotechnology Index. The driving factors for value growth primarily come from the proof of concept of the delivery system and the proof of concept of rare diseases and common disease indications and the approval to enter the market. Novartis spent USD 9.7 billion to acquire PCSK9- targeted RNAi drug, marking a huge room for future expansion of this type of drugs in the treatment of major diseases and common diseases. The current clinical pipeline is basically more mature with rare disease treatment, and the application for common diseases and tumors is constantly expanding. With regard to targeted organs, the liver has become the most technologically mature and competitive target organ.

The future development efforts for RNAi therapeutics- related technologies and clinical applications can be focused on ① breaking through the limitations of existing delivery technologies and selecting more effective and safer technologies for more tissues and organs, with carriers to achieve a wider security window and broader application scenarios; ② expanding the existing delivery technology into the fields of polypeptide nanoparticles and polypeptide ligand targeting; ③ optimizing target selection for chronic diseases to improve specific and powerful targets, target the tumor microenvironment, and improve the activity of anti- tumor drugs through strategies such as dual targets and multiple targets, coupled drugs, and combination therapies; ④ expanding into the field of non-liver and non-rare diseases to meet the individualized medical needs and treatments; and ⑤ strengthening the protection of intellectual property rights to improve the quality of patent applications and expand the citation rate. In addition, the transition from domestic patent applications to PCT applications should be accelerated as much as possible. The quality of international intellectual property applications and the protection of independent innovation technology platforms and products should be improved.

《2.2 Interpretations for three key engineering development fronts》

2.2 Interpretations for three key engineering development fronts

2.2.1 Clinical translational application of brain–computer interface technology

The brain–computer interface (BCI) directly links the human or animal brain (or brain cell culture) and external equipment. The BCI can be a new information transmission channel through which information can bypass the original muscle and peripheral nerve pathways. It can achieve the unidirectional/bidirectional information exchange between brain and external equipment. Human brain activity was first recorded by electroencephalography (EEG) in 1924. Brain–computer interface technology was based on EEG and originated in the 1970s. The first neuroprosthetic device implanted in the human body appeared in the mid-1990s. Modern BCI technology covers the monitoring, regulation, and functional replacement of the nervous system and fulfills the imagination of controlling objects and memory storage and transplantation.

Based on the technical process, BCI technology can be divided into acquisition, processing, control, and feedback. The critical technical problems that need to be solved primarily include BCI equipment with high throughput, high signal- to-noise ratio, high temporal and spatial resolution, and high security in the acquisition, including electrode and chip. The critical point of processing lies in neural decoding. However, significant differences in individual EEG signals, brain regions, and behaviors are observed. EEG processing algorithms, clinical cognition of EEG data, and data volume are the bottlenecks in developing neural decoding. At present, the control part is relatively mature in the overall technical process. Feedback is the key to achieve bidirectional BCI, and the corresponding relationship between neural coding and function needs further verification.

At present, the research hotspot areas of BCI technology in clinical translational application mainly include:

1)  Diagnosis and treatment of limb movement disorders: The auxiliary BCI obtains patient’s movement intention through equipment and achieves the control of external equipment such as prosthesis or exoskeleton control. The rehabilitation BCI directly acts on the brain to perform repetitive feedback stimulation, enhance the connection between neuronal synapses, and promote nerve repair.

2)  Monitoring, diagnosis, and treatment of consciousness and cognitive disorders: The level of consciousness assessment and prognostic judgment conducted by monitoring the EEG of patients with consciousness and cognitive disorders and the diagnosis and stimulation treatments on early symptoms of cognitive disorders are a hot application and research direction in the consciousness and cognition obstacle field.

3)  Diagnosis and treatment of mental illness: By extracting the characteristics of EEG signals, it is possible to recognize a variety of emotions and then assist the pathogenesis research and conduct targeted treatment on depression, anxiety, and other mental illnesses. Neurofeedback training based on the BCI is also a feasible rehabilitation treatment method for mental illnesses.

4)   Diagnosis and treatment of sensory defects: Through the decoding and encoding of sensory information, BCI technology can be applied to nerve stimulation of sensory defects such as hearing, vision, and touch to help patients recover part of their sensations.

5)   Diagnosis and treatment of epilepsy: In the diagnosis of epilepsy, electrophysiological abnormalities have always been the gold standard for clinical diagnosis. Applying electrical stimulation, magnetic stimulation, and closed-loop control technique to treat epilepsy is also widely used in clinical practice.

Scientific research institutes mostly dominate the BCI industry at home and abroad. Given the technical, ethical, safety, and other restrictions, their research routes are primarily non-invasive BCI, and invasive BCI are limited to applications in medical and health scenarios. The number of research institutions and research investments involved in non-invasive BCI is low. Given the high R&D costs, lack of professional talents, and unclear profit models, significantly fewer companies are involved in the BCI industry compared with other AI fields. However, Neuralink’s press conference, Ali Dharma Academy’s technology trend forecast, and domestic Neuracle’s more than CNY 100 million round of financing led by Sequoia Capital pay considerable attention to domestic and foreign capital in this field. Regarding application landing, the certification and supervision of related products at home and abroad are still in its infancy. In the past few years, the FDA has only reviewed several clinical application products of BrainGate, Ceribell, and NeuroPace.

In the future, the BCI field will gradually form an open ecosystem. Market demand-oriented application BCI companies are expected to expand the industrial scale rapidly by further mining demand scenarios. In addition, many companies specializing in implantable chips and application of APP development will also emerge. Similar to the current research status at home and abroad, the application of BCI to medical rehabilitation will continue to be the largest market with the fastest short-term growth in the future. As for the most imaginative advanced human-computer interaction field, given the technical limitations and bottlenecks in the development of cognitive science, producing mature general- purpose products or achieving large-scale commercialization in the short term are difficult.

For medical and health scenarios, including sleep disorders, Parkinson’s disease, Alzheimer’s disease, hemiplegia, epilepsy, depression, and other diseases, given the high willingness of users to try, many parties, including the capital, hospitals, and companies, believe that they will be the first to usher in growth. According to calculations of relevant institutions, the current market size of China’s BCI equipment is at the billion levels. By 2040, the total market size of China’s BCI industry will exceed CNY 100 billion, and the market growth rate is significantly higher than the global growth.

The major producing countries of core patents of BCI technology are China, the USA, South Korea, Japan, and India (Table 2.2.1). From the perspective of the cooperation network between the countries where the core patents are produced, the USA, Germany, and the Netherlands have cooperated closely (Figure 2.2.1).

The top institutions with regard to core patent output are Tianjin University, South China University of Technology, and Xi’an Jiaotong University (Table 2.2.2). Moreover, a cooperative relationship between Institute of Biomedical Engineering of Chinese Academy of Medical Sciences and Tsinghua Shenzhen International Graduate School is observed (Figure 2.2.2).

2.2.2 Genetically engineered organ xenotransplantation technology

Organ xenotransplantation is a process of transplantation of animal-derived organs and human-derived organs grown from xenogeneic materials in vitro into human beings. With the success of surgical operations, the continuous development of immunosuppressants and the improvement of the success rate of transplantation, organ transplantation has gradually become the preferred method for the treatment of end-stage diseases, but donors are limited. Therefore, organ xenotransplantation has become a research hotspot. Since the world’s first organ xenotransplant in 1905, xenotransplantation has gone through several stages

《Table 2.2.1》

Table 2.2.1 Countries with the greatest output of core patents on “clinical translational application of brain–computer interface technology”

《Table 2.2.2》

Table 2.2.2 Institutions with the greatest output of core patents on “clinical translational application of brain–computer interface technology”

《Figure 2.2.1》

Figure 2.2.1 Collaboration network among major countries in the engineering development front of “clinical translational application of brain–computer interface technology”

《Figure 2.2.2》

Figure 2.2.2 Collaboration network among major institutions in the engineering development front of “clinical translational application of brain–computer interface technology”

of development: In the 1980s, the primary challenge was overcoming hyperacute rejection. Then, delayed immune rejection of transplanted organs was concerned in the 1990s. The production of GTKO pig in the 21st century has completely changed the historical course of organ xenotransplantation. The innovation of gene editing and immunosuppressive agents has made breakthroughs in the survival of pig organ xenografts, and the potential of clinical application has become clear.

At present, the development of genetically engineered organ xenotransplantation must solve the following key technical problems: ① exploration of gene editing sites and strategy within donor pigs, ② stable breeding and reproduction of multi-gene editing donor pigs, ③ endogenous retrovirus knockout and SPF environmental feeding of donor pigs, ④ development of new immunosuppressants to inhibit xenogeneic rejection, and suitable immunosuppressive regimens for clinical application, ⑤ establishment of evaluation criteria for biosafety and efficacy of xenografts and management system for xenotransplantation process, ⑥ establishment of clinical indications for patients undergoing clinical xenotransplantation, ⑦ the standardization of tracking, supervision and management of transplant patients, and ⑧ social recognition and ethical review system. In addition, the hot sub-areas of international research about organ xenotransplantation include:

1)   Tissue and organ engineering: cell culture technology is used to artificially control cell differentiation, proliferation, and growth into needed tissues and organs in vitro, and the engineered mass production of tissues and organs can be used to repair and replace the lost tissues and organs in vivo to meet the needs of clinical and rehabilitation. At present, the USA is in the first place in the field of tissue and organ engineering in basic research and applied research. China has not yet formed competitiveness in tissue and organ engineering industry. However, considering that tissue and organ engineering is an emerging industry with rapid progress in the recent 3–5 years, there is not much gap between China and the international market.

2)   Human-pig chimeric organs: using human skin tissue to induce the generation of pluripotent stem cells by transplanting them into early pig embryos, making adult pig chimeric embryos, and growing human organs on pigs for human organ transplantation is the latest research direction in the field of organ xenotransplantation. Since scientists created chimeric embryos for the first time, the study of chimeric embryos for humans and pigs has faced great ethical controversy, and it is still far from the ultimate goal of creating organs for transplantation.

3)   Humanized pig organs: pigs have become an ideal donor for organ xenotransplantation because of their organ size, physiological structure, and metabolic characteristics similar to those of human beings and their convenience in breeding and rapid growth. Pig organs have been genetically engineered to “disguise” themselves as human organs; thus, the human immune system cannot attack these humanized pig organs. In recent years, great breakthroughs have been made in the preclinical research. For example, CRISPR/ Cas9 gene editing has made the process of humanization of pig organs efficient and simple. The novel T cell activation costimulatory signal blockade has a significant effect on xenogeneic rejection. The survival of pig-derived xenografts in non-human primates is increasing, and clinical applications are getting closer.

Organ shortage is a worldwide problem; the USA waits for nearly 110 000 transplant patients every year and receives less than 40 000 transplant patients every year. More than 300 000 patients need organ transplants in China every year, and only 10 000 patients receive transplants annually; thus, the market demand is huge. In 2019, Massachusetts General Hospital performed the world’s first gene-edited pig-to-human skin xenotransplant. In 2020, the US FDA approved genetically modified pigs for use in food and medical products. In 2021, Science published 11 cutting-edge medical problems in the world, one of which was xenotransplantation. In the same year, Miromatrix Medical, the world’s first pig organ transplant company, went public on NASDAQ. In the USA, the clinical application of pig kidney and heart xenotransplantation can be conducted in the next 2 or 5 years. China ranks first in this field, and the cultivation of GTKO and PERV-KO pigs is led by Chinese scientists. The international symposium on clinical norms for organ xenotransplantation was held in China two times. Chinese scientists have repeatedly broken the survival record for preclinical pig liver xenotransplantation. At present, China is facing great opportunities and challenges; thus, accelerating the transformation of xenotransplantation from basic research to clinical application is necessary.

At present, 314 core patents have been applied on the frontier direction of “genetically engineered organ xenotransplantation technology”. The USA, China, South Korea, the UK, and Germany are the top five countries with the most patents in force. The patents applied by Chinese authors accounted for 20.70% of the total patents, and the proportion of the number of patents is large (Table 2.2.3). China has become one of the key countries researching on this aspect of engineering development. From cooperation network among countries producing core patents, the USA has close cooperation with Switzerland, the UK, Canada, Germany, and China (Figure 2.2.3). Dana-Farber Cancer Institute, Abbott Laboratories, and Yunnan Agricultural University ranked first with regard to the output of core patents (Table 2.2.4). Moreover, partnerships between Konkuk University and the University of Missouri, Dana-Farber Cancer Institute and Novartis AG, and Roche Pharmaceuticals and Genentech, Inc., are found (Figure 2.2.4).

2.2.3 Tumor neoantigen vaccines

Vaccines targeting neoantigens represent a novel and potential immunotherapy that induces specific immune responses to eliminate tumor cells. Neoantigens can arise through a variety of “non-synonymous mutation” events, which exist only in cancer cells but not in normal cells, with

Table 2.2.3 Countries with the greatest output of core patents on “genetically engineered organ xenotransplantation technology”

Table 2.2.4 Institutions with the greatest output of core patents on “genetically engineered organ xenotransplantation technology”

the numbers and types of mutations varying by cancer types. These events include point mutations, insertions or deletions (indels), and gene fusions. Viral proteins can be considered as an alternative class of neoantigens derived from viruses in tumors. At present, neoantigen vaccines include four major types, that is, immune cell-based vaccines (e.g., dendritic cells or T-cell receptor-engineered T cells), peptide vaccines, viral vector-based vaccines, and nucleic acid-based vaccines (e.g., DNA or RNA vaccines). Traditional therapeutic vaccination strategies usually focus on self-antigens that are abnormally expressed or overexpressed in tumors, also termed as tumor- associated antigens (TAAs). For example, Provenge is a traditional therapeutic vaccine for patients with advanced prostate cancer, which is approved by the USA FDA. With the rapid development of sequencing technology in recent years, genetic variations have been continuously identified, and therapeutic vaccines targeting neoantigens have achieved major breakthroughs in the treatment of patients with malignant tumors. Vaccines based on neoantigens rather than traditional TAAs have several advantages. First, neoantigens are exclusively expressed by tumors cells; therefore, they can elicit tumor-specific T cell responses, thereby preventing “off- target” damage to nonmalignant tissues. Second, neoantigens are de novo antigens derived from somatic mutations, which can circumvent T cell central tolerance of self-antigens and thus induce specific immune responses to tumors. In addition, the specific T cell responses induced by neoantigen vaccines can persist and provide post-treatment immunological memory, thereby increasing the possibility of long-term protection against disease recurrence. In recent clinical trials, neoantigen vaccines significantly prolong the disease-free survival time, bringing new hope to patients with malignant tumors. The diversity of immune responses can be induced by neoantigens, with the numbers and types of neoantigens varying by cancer types, which lead to personalized approach to neoantigen vaccines. The demand for “individualization” of neoantigen vaccines poses new challenges to bioinformatics and plays a positive role in promoting contemporary medical innovation.

The development and application of personalized neoantigen vaccines usually include: ① identification of somatic mutations through high-throughput sequencing and analysis based on tumor and non-malignant tissues from cancer patients; ② prediction of neoantigen immunogenicity by available algorithms; ③ validation of highly immunogenic neoantigens through in vitro immunological experiments (e.g., ELISPOT); ④ preparation of neoantigen vaccines; and ⑤ infusion of vaccines and monitoring of specific anti-tumor efficacies. The main technical issues that need to be solved include the accuracy of somatic mutation and neoantigen screening; the large-scale, cost-effective, and standardized production of high-quality neoantigen vaccines; the interpretation of the mechanisms and biological effects of neoantigen vaccines; the optimal vaccine delivery vehicles; the suitable vaccine format; and the application of immunotherapy and combination therapy based on neoantigen vaccines. The current hotspots in the field of neoantigen vaccine research include the following:

1)  Identification of the optimal neoantigen targets: including mapping genetic alterations in the tumor genome using whole genome sequencing (WGS) or whole exome sequencing (WES) data from matched tumor and normal DNA, and RNA sequencing (RNA-seq) from tumor RNA; improving the accuracy of neoantigen immunogenicity prediction through integrating multiple algorithms, such as affinity, somatic mutation VAF, gene expression level, and clonality; the integration of HLA-binding peptide data achieved by liquid chromatography-tandem mass spectrometry to increase the accuracy and specificity of neoantigen screening.

2)  Vaccine formats for the delivery of neoantigens: synthetic peptides, mRNAs, DNA plasmids, viral vectors (e.g., adenovirus and vaccinia), engineered attenuated bacterial vectors (e.g., Salmonella and Listeria), and ex vivo neoantigen-loaded DCs. The advantages of peptide vaccines are automatic synthesis, transient activity, and complete degradation. The mRNA- based vaccines have inherent adjuvant function via TLR7, TLR8, and TLR3 signaling. The DNA-based vaccines are cost effective, and they have straightforward manufacturing and inherent adjuvant activity driven by TLR9. The advantages of viral vectors are their strong immunostimulatory activity and extensive clinical experience in the field of infectious diseases. Engineered attenuated bacteria vectors have strong immunostimulatory activity, and they could be combined with plasmid DNA. DCs can be loaded with various neoantigen formats, and they have been proven clinical efficacy.

3)   Neoantigen-specific T-cell-receptor-engineered T cells (TCR-T), including isolating neoantigen-specific T cells from tumor-infiltrating lymphocytes (TILs), analyzing the V(D) J sequences of TCR, constructing the TCR structure, and transferring into patients’ T cells, to establish TCR-T cells.

4)   Determining the suitable clinical setting for neoantigen vaccine-based therapy. Therapeutic vaccines mostly work particularly well in the adjuvant or minimal residual disease settings, where the tumor load is low and immune- suppressive mechanisms are not firmly established.

5)   Format of neoantigen vaccine-based therapy, including the combination of other immunotherapy, to expand the efficacy of neoantigen vaccines, such as the use of immune checkpoint inhibitors (PD1/PD-L1, CTLA4, LAG-3, TIM-3, IDO, etc.), stimulating molecules (OX40, GITR, CD137, etc.), or T-cell-activating factors (GM-CSF, IL-2, etc.).

The market demand for tumor neoantigen vaccines is enormous. With the trends and technologies of the digital age, such as big data science, cloud and high-performance computing, and machine learning tools, the neoantigen predictive algorithms will be continuously improved. TCR repertoire analysis, high-throughput single-cell sequencing, and circulating tumor DNA detection will provide high-resolution analysis of tumors, the microenvironment, and immunity to determine suitable time with the neoantigen vaccine treatments. Computational inference of the phenotype and functional status of infiltrating cells from transcriptome data may support the selection of combination treatments. Therefore, personalized neoantigen vaccines can be a universally applicable therapy irrespective of cancer type.

The number of core patent disclosures of “tumor neoantigen vaccines” is increasing every year. China, USA, Japan, Germany, and South Korea are the top five countries with the most patents in force. China has become one of the key countries researching on this aspect of engineering development with the patents applied by Chinese authors accounted for 35.01% of the total patents (Table 2.2.5). As shown in the cooperation network of patent-producing countries (Figure 2.2.5), the USA and Switzerland cooperate more closely. The top institutions with regard to the number of core patent outputs are Swiss Roche Pharmaceuticals, US Department of Health and Human Services, and University of Pennsylvania (Table 2.2.6). Moreover, a cooperative relationship is observed between Sloan-Kettering Cancer Center and US Department of Health and Human Services (Figure 2.2.6).

《Table 2.2.5》

Table 2.2.5 Countries with the greatest output of core patents on “tumor neoantigen vaccines”

《Table 2.2.6》

Table 2.2.6 Institutions with the greatest output of core patents on “tumor neoantigen vaccines”