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200+ Biotechnology Research Topics: Let’s Shape the Future

In the dynamic landscape of scientific exploration, biotechnology stands at the forefront, revolutionizing the way we approach healthcare, agriculture, and environmental sustainability. This interdisciplinary field encompasses a vast array of research topics that hold the potential to reshape our world. 

In this blog post, we will delve into the realm of biotechnology research topics, understanding their significance and exploring the diverse avenues that researchers are actively investigating.

Overview of Biotechnology Research

Table of Contents

Biotechnology, at its core, involves the application of biological systems, organisms, or derivatives to develop technologies and products for the benefit of humanity. 

The scope of biotechnology research is broad, covering areas such as genetic engineering, biomedical engineering, environmental biotechnology, and industrial biotechnology. Its interdisciplinary nature makes it a melting pot of ideas and innovations, pushing the boundaries of what is possible.

How to Select The Best Biotechnology Research Topics?

• Identify Your Interests

Start by reflecting on your own interests within the broad field of biotechnology. What aspects of biotechnology excite you the most? Identifying your passion will make the research process more engaging.

• Stay Informed About Current Trends

Keep up with the latest developments and trends in biotechnology. Subscribe to scientific journals, attend conferences, and follow reputable websites to stay informed about cutting-edge research. This will help you identify gaps in knowledge or areas where advancements are needed.

• Consider Societal Impact

Evaluate the potential societal impact of your chosen research topic. How does it contribute to solving real-world problems? Biotechnology has applications in healthcare, agriculture, environmental conservation, and more. Choose a topic that aligns with the broader goal of improving quality of life or addressing global challenges.

• Assess Feasibility and Resources

Evaluate the feasibility of your research topic. Consider the availability of resources, including laboratory equipment, funding, and expertise. A well-defined and achievable research plan will increase the likelihood of successful outcomes.

• Explore Innovation Opportunities

Look for opportunities to contribute to innovation within the field. Consider topics that push the boundaries of current knowledge, introduce novel methodologies, or explore interdisciplinary approaches. Innovation often leads to groundbreaking discoveries.

• Consult with Mentors and Peers

Seek guidance from mentors, professors, or colleagues who have expertise in biotechnology. Discuss your research interests with them and gather insights. They can provide valuable advice on the feasibility and significance of your chosen topic.

• Balance Specificity and Breadth

Strike a balance between biotechnology research topics that are specific enough to address a particular aspect of biotechnology and broad enough to allow for meaningful research. A topic that is too narrow may limit your research scope, while one that is too broad may lack focus.

• Consider Ethical Implications

Be mindful of the ethical implications of your research. Biotechnology, especially areas like genetic engineering, can raise ethical concerns. Ensure that your chosen topic aligns with ethical standards and consider how your research may impact society.

• Evaluate Industry Relevance

Consider the relevance of your research topic to the biotechnology industry. Industry-relevant research has the potential for practical applications and may attract funding and collaboration opportunities.

• Stay Flexible and Open-Minded

Be open to refining or adjusting your research topic as you delve deeper into the literature and gather more information. Flexibility is key to adapting to new insights and developments in the field.

200+ Biotechnology Research Topics: Category-Wise

Genetic engineering.

• CRISPR-Cas9: Recent Advances and Applications
• Gene Editing for Therapeutic Purposes: Opportunities and Challenges
• Precision Medicine and Personalized Genomic Therapies
• Genome Sequencing Technologies: Current State and Future Prospects
• Synthetic Biology: Engineering New Life Forms
• Genetic Modification of Crops for Improved Yield and Resistance
• Ethical Considerations in Human Genetic Engineering
• Gene Therapy for Neurological Disorders
• Epigenetics: Understanding the Role of Gene Regulation
• CRISPR in Agriculture: Enhancing Crop Traits

Biomedical Engineering

• Tissue Engineering: Creating Organs in the Lab
• 3D Printing in Biomedical Applications
• Advances in Drug Delivery Systems
• Nanotechnology in Medicine: Theranostic Approaches
• Bioinformatics and Computational Biology in Biomedicine
• Wearable Biomedical Devices for Health Monitoring
• Stem Cell Research and Regenerative Medicine
• Precision Oncology: Tailoring Cancer Treatments
• Biomaterials for Biomedical Applications
• Biomechanics in Biomedical Engineering

Environmental Biotechnology

• Bioremediation of Polluted Environments
• Waste-to-Energy Technologies: Turning Trash into Power
• Sustainable Agriculture Practices Using Biotechnology
• Bioaugmentation in Wastewater Treatment
• Microbial Fuel Cells: Harnessing Microorganisms for Energy
• Biotechnology in Conservation Biology
• Phytoremediation: Plants as Environmental Cleanup Agents
• Aquaponics: Integration of Aquaculture and Hydroponics
• Biodiversity Monitoring Using DNA Barcoding
• Algal Biofuels: A Sustainable Energy Source

Industrial Biotechnology

• Enzyme Engineering for Industrial Applications
• Bioprocessing and Bio-manufacturing Innovations
• Industrial Applications of Microbial Biotechnology
• Bio-based Materials: Eco-friendly Alternatives
• Synthetic Biology for Industrial Processes
• Metabolic Engineering for Chemical Production
• Industrial Fermentation: Optimization and Scale-up
• Biocatalysis in Pharmaceutical Industry
• Advanced Bioprocess Monitoring and Control
• Green Chemistry: Sustainable Practices in Industry

Emerging Trends in Biotechnology

• CRISPR-Based Diagnostics: A New Era in Disease Detection
• Neurobiotechnology: Advancements in Brain-Computer Interfaces
• Advances in Nanotechnology for Healthcare
• Computational Biology: Modeling Biological Systems
• Organoids: Miniature Organs for Drug Testing
• Genome Editing in Non-Human Organisms
• Biotechnology and the Internet of Things (IoT)
• Exosome-based Therapeutics: Potential Applications
• Biohybrid Systems: Integrating Living and Artificial Components
• Metagenomics: Exploring Microbial Communities

Ethical and Social Implications

• Ethical Considerations in CRISPR-Based Gene Editing
• Privacy Concerns in Personal Genomic Data Sharing
• Biotechnology and Social Equity: Bridging the Gap
• Dual-Use Dilemmas in Biotechnological Research
• Informed Consent in Genetic Testing and Research
• Accessibility of Biotechnological Therapies: Global Perspectives
• Human Enhancement Technologies: Ethical Perspectives
• Biotechnology and Cultural Perspectives on Genetic Modification
• Social Impact Assessment of Biotechnological Interventions
• Intellectual Property Rights in Biotechnology

Computational Biology and Bioinformatics

• Machine Learning in Biomedical Data Analysis
• Network Biology: Understanding Biological Systems
• Structural Bioinformatics: Predicting Protein Structures
• Data Mining in Genomics and Proteomics
• Systems Biology Approaches in Biotechnology
• Comparative Genomics: Evolutionary Insights
• Bioinformatics Tools for Drug Discovery
• Cloud Computing in Biomedical Research
• Artificial Intelligence in Diagnostics and Treatment
• Computational Approaches to Vaccine Design

Health and Medicine

• Vaccines and Immunotherapy: Advancements in Disease Prevention
• CRISPR-Based Therapies for Genetic Disorders
• Infectious Disease Diagnostics Using Biotechnology
• Telemedicine and Biotechnology Integration
• Biotechnology in Rare Disease Research
• Gut Microbiome and Human Health
• Precision Nutrition: Personalized Diets Using Biotechnology
• Biotechnology Approaches to Combat Antibiotic Resistance
• Point-of-Care Diagnostics for Global Health
• Biotechnology in Aging Research and Longevity

Agricultural Biotechnology

• CRISPR and Gene Editing in Crop Improvement
• Precision Agriculture: Integrating Technology for Crop Management
• Biotechnology Solutions for Food Security
• RNA Interference in Pest Control
• Vertical Farming and Biotechnology
• Plant-Microbe Interactions for Sustainable Agriculture
• Biofortification: Enhancing Nutritional Content in Crops
• Smart Farming Technologies and Biotechnology
• Precision Livestock Farming Using Biotechnological Tools
• Drought-Tolerant Crops: Biotechnological Approaches

Biotechnology and Education

• Integrating Biotechnology into STEM Education
• Virtual Labs in Biotechnology Teaching
• Biotechnology Outreach Programs for Schools
• Online Courses in Biotechnology: Accessibility and Quality
• Hands-on Biotechnology Experiments for Students
• Bioethics Education in Biotechnology Programs
• Role of Internships in Biotechnology Education
• Collaborative Learning in Biotechnology Classrooms
• Biotechnology Education for Non-Science Majors
• Addressing Gender Disparities in Biotechnology Education

Funding and Policy

• Government Funding Initiatives for Biotechnology Research
• Private Sector Investment in Biotechnology Ventures
• Impact of Intellectual Property Policies on Biotechnology
• Ethical Guidelines for Biotechnological Research
• Public-Private Partnerships in Biotechnology
• Regulatory Frameworks for Gene Editing Technologies
• Biotechnology and Global Health Policy
• Biotechnology Diplomacy: International Collaboration
• Funding Challenges in Biotechnology Startups
• Role of Nonprofit Organizations in Biotechnological Research

Biotechnology and the Environment

• Biotechnology for Air Pollution Control
• Microbial Sensors for Environmental Monitoring
• Remote Sensing in Environmental Biotechnology
• Climate Change Mitigation Using Biotechnology
• Circular Economy and Biotechnological Innovations
• Marine Biotechnology for Ocean Conservation
• Bio-inspired Design for Environmental Solutions
• Ecological Restoration Using Biotechnological Approaches
• Impact of Biotechnology on Biodiversity
• Biotechnology and Sustainable Urban Development

Biosecurity and Biosafety

• Biosecurity Measures in Biotechnology Laboratories
• Dual-Use Research and Ethical Considerations
• Global Collaboration for Biosafety in Biotechnology
• Security Risks in Gene Editing Technologies
• Surveillance Technologies in Biotechnological Research
• Biosecurity Education for Biotechnology Professionals
• Risk Assessment in Biotechnology Research
• Bioethics in Biodefense Research
• Biotechnology and National Security
• Public Awareness and Biosecurity in Biotechnology

Industry Applications

• Biotechnology in the Pharmaceutical Industry
• Bioprocessing Innovations for Drug Production
• Industrial Enzymes and Their Applications
• Biotechnology in Food and Beverage Production
• Applications of Synthetic Biology in Industry
• Biotechnology in Textile Manufacturing
• Cosmetic and Personal Care Biotechnology
• Biotechnological Approaches in Renewable Energy
• Advanced Materials Production Using Biotechnology
• Biotechnology in the Automotive Industry

Miscellaneous Topics

• DNA Barcoding in Species Identification
• Bioart: The Intersection of Biology and Art
• Biotechnology in Forensic Science
• Using Biotechnology to Preserve Cultural Heritage
• Biohacking: DIY Biology and Citizen Science
• Microbiome Engineering for Human Health
• Environmental DNA (eDNA) for Biodiversity Monitoring
• Biotechnology and Astrobiology: Searching for Life Beyond Earth
• Biotechnology and Sports Science
• Biotechnology and the Future of Space Exploration

Challenges and Ethical Considerations in Biotechnology Research

As biotechnology continues to advance, it brings forth a set of challenges and ethical considerations. Biosecurity concerns, especially in the context of gene editing technologies, raise questions about the responsible use of powerful tools like CRISPR. 

Ethical implications of genetic manipulation, such as the creation of designer babies, demand careful consideration and international collaboration to establish guidelines and regulations. 

Moreover, the environmental and social impact of biotechnological interventions must be thoroughly assessed to ensure responsible and sustainable practices.

Funding and Resources for Biotechnology Research

The pursuit of biotechnology research topics requires substantial funding and resources. Government grants and funding agencies play a pivotal role in supporting research initiatives. 

Simultaneously, the private sector, including biotechnology companies and venture capitalists, invest in promising projects. Collaboration and partnerships between academia, industry, and nonprofit organizations further amplify the impact of biotechnological research.

Future Prospects of Biotechnology Research

As we look to the future, the integration of biotechnology with other scientific disciplines holds immense potential. Collaborations with fields like artificial intelligence, materials science, and robotics may lead to unprecedented breakthroughs. 

The development of innovative technologies and their application to global health and sustainability challenges will likely shape the future of biotechnology.

In conclusion, biotechnology research is a dynamic and transformative force with the potential to revolutionize multiple facets of our lives. The exploration of diverse biotechnology research topics, from genetic engineering to emerging trends like synthetic biology and nanobiotechnology, highlights the breadth of possibilities within this field. 

However, researchers must navigate challenges and ethical considerations to ensure that biotechnological advancements are used responsibly for the betterment of society. 

With continued funding, collaboration, and a commitment to ethical practices, the future of biotechnology research holds exciting promise, propelling us towards a more sustainable and technologically advanced world.

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The future is bright, the future is biotechnology

* E-mail: [email protected]

Affiliation Public Library of Science, San Francisco, California, United States of America and Cambridge, United Kingdom

• Richard Hodge, 
• on behalf of the PLOS Biology staff editors

Published: April 28, 2023

• https://doi.org/10.1371/journal.pbio.3002135
• Reader Comments

As PLOS Biology celebrates its 20 th anniversary, our April issue focuses on biotechnology with articles covering different aspects of the field, from genome editing to synthetic biology. With them, we emphasize our interest in expanding our presence in biotechnology research.

Citation: Hodge R, on behalf of the PLOS Biology staff editors (2023) The future is bright, the future is biotechnology. PLoS Biol 21(4): e3002135. https://doi.org/10.1371/journal.pbio.3002135

Copyright: © 2023 Hodge, on behalf of the PLOS Biology staff editors. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The authors received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

The PLOS Biology Staff Editors are Ines Alvarez-Garcia, Joanna Clarke, RichardHodge, Paula Jauregui, Nonia Pariente, Roland Roberts, and Lucas Smith.

This article is part of the PLOS Biology 20th Anniversary Collection.

Biotechnology is a revolutionary branch of science at the forefront of research and innovation that has advanced rapidly in recent years. It is a broad discipline, in which organisms or biological processes are exploited to develop new technologies that have the potential to transform the way we live and work, as well as to boost sustainability and industrial productivity. The new tools and products being generated have a wide range of applications across various sectors, including medicine, agriculture, energy, manufacturing and food.

PLOS Biology has traditionally published research reporting significant advances across a wide range of biological disciplines. However, our scope must continue to evolve as biology increasingly becomes more and more applied, generating technologies with potentially game-changing therapeutic and environmental impact. To that end, we recently published a collection of magazine articles focused on ideas for green biotechnologies that could have an important role in a sustainable future [ 1 ], including how to harness microbial photosynthesis to directly generate electricity [ 2 ] and using microbes to develop carbon “sinks” in the mining industry [ 3 ]. Moreover, throughout this anniversary year we are publishing Perspective articles that take stock of the past 20 years of biological research in a specific field and look forward to what is to come in the next 20 years [ 4 ]; in this issue, these Perspectives focus on different aspects of the broad biotechnology field—synthetic biology [ 5 ] and the use of lipid nanoparticles (LNPs) for the delivery of therapeutics [ 6 ].

One fast moving area within biotechnology is gene editing therapy, which involves the alteration of DNA to treat or prevent disease using techniques such as CRISPR-Cas9 and base editors that enable precise genetic modifications to be made. This approach shows great promise for treating a variety of genetic diseases. Excitingly, promising phase I results of the first in vivo genome editing clinical trial to treat several liver-related diseases were reported at the recent Keystone Symposium on Precision Genome Engineering. This issue of PLOS Biology includes an Essay from Porto and Komor that focuses on the clinical applications of base editor technology [ 7 ], which could enable chronic diseases to be treated with a ‘one-and-done’ therapy, and a Perspective from Hamilton and colleagues that outlines the advances in the development of LNPs for the delivery of nucleic acid-based therapeutics [ 6 ]. LNPs are commonly used as vehicles for the delivery of such therapeutics because they have a low immunogenicity and can be manufactured at scale. However, expanding the toolbox of delivery platforms for these novel therapeutics will be critical to realise their full clinical potential.

Synthetic biology is also a rapidly growing area, whereby artificial or existing biological systems are designed to produce products or enhance cellular function. By using CRISPR to edit genes involved in metabolic pathways, researchers can create organisms that produce valuable compounds such as biofuels, drugs, and industrial chemicals. In their Perspective, Kitano and colleagues take stock of the technological advances that have propelled the “design-build-test-learn” cycle methodology forward in synthetic biology, as well as focusing on how machine-learning approaches can remove the bottlenecks in these pipelines [ 5 ].

While the potential of these technologies is vast, there are also concerns about their safety and ethical implications. Gene editing, in particular, raises ethical concerns, as it could be used to create so-called “designer babies” with specific traits or to enhance physical or mental capabilities. There are also concerns about the unintended consequences of gene editing, such as off-target effects that could cause unintended harm. These technologies can be improved by better understanding the interplay between editing tools and DNA repair pathways, and it will be essential for scientists and policymakers to be cautious and work together to establish guidelines and regulations for their use, as outlined at the recent International Summit on Human Genome Editing .

Basic research has also benefitted from biotechnological developments. For instance, methodological developments in super-resolution microscopy offer researchers the ability to image cells at exquisite detail and answer previously inaccessible research questions. Sequencing technologies such as Nanopore sequencers are revolutionising the ability to sequence long DNA/RNA reads in real time and in the field. Great strides have also been made in the development of analysis software for structural biology purposes, such as sub-tomogram averaging for cryo-EM [ 8 ]. The rate of scientific discovery is now at an unprecedented level in this age of big data as a result of these huge technological leaps.

The past few years has also seen the launch of AI tools such as ChatGPT. While these tools are increasingly being used to help write students homework or to improve the text of scientific papers, generative AI tools hold the potential to transform research and development in the biotechnology industry. The recently developed language model ProGen can generate and then predict function in protein sequences [ 9 ], and these models can also be used to find therapeutically relevant compounds for drug discovery. Protein structure prediction programs, such as AlphaFold [ 10 ] and RosettaFold, have revolutionized structural biology and can be used for a myriad of purposes. We have recently published several papers that have utilized AlphaFold models to develop methods that determine the structural context of post-translational modifications [ 11 ] and predict autophagy-related motifs in proteins [ 12 ].

The future of biotechnology is clearly very promising and we look forward to being part of the dissemination of these important new developments. Open access science sits at the core of our mission and the publication of these novel technologies in PLOS Biology can help their widespread adoption and ensure global access. As we look forward during this year of celebration, we are excited that biotechnology research will continue to grow and become a central part of the journal. The future is bright and the future is very much biotechnology.

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Biotechnology Research Paper Topics

This collection of biotechnology research paper topics provides the list of 10 potential topics for research papers and overviews the history of biotechnology.

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Get 10% off with 24start discount code, 1. animal breeding: genetic methods.

Modern animal breeding relies on scientific methods to control production of domesticated animals, both livestock and pets, which exhibit desired physical and behavioral traits. Genetic technology aids animal breeders to attain nutritional, medical, recreational, and fashion standards demanded by consumers for animal products including meat, milk, eggs, leather, wool, and pharmaceuticals. Animals are also genetically designed to meet labor and sporting requirements for speed and endurance, conformation and beauty ideals to win show competitions, and intelligence levels to perform obediently at tasks such as herding, hunting, and tracking. By the late twentieth century, genetics and mathematical models were appropriated to identify the potential of immature animals. DNA markers indicate how young animals will mature, saving breeders money by not investing in animals lacking genetic promise. Scientists also successfully transplanted sperm-producing stem cells with the goal of restoring fertility to barren breeding animals. At the National Animal Disease Center in Ames, Iowa, researchers created a gene-based test, which uses a cloned gene of the organism that causes Johne’s disease in cattle in order to detect that disease to avert epidemics. Researchers also began mapping the dog genome and developing molecular techniques to evaluate canine chromosomes in the Quantitative Trait Loci (QTL). Bioinformatics incorporates computers to analyze genetic material. Some tests were developed to diagnose many of several hundred genetic canine diseases including hip dysplasia and progressive retinal atrophy (PRA). A few breed organizations modified standards to discourage breeding of genetically flawed animals and promote heterozygosity.

2. Antibacterial Chemotherapy

In the early years of the twentieth century, the search for agents that would be effective against internal infections proceeded along two main routes. The first was a search for naturally occurring substances that were effective against microorganisms (antibiosis). The second was a search for chemicals that would have the same effect (chemotherapy). Despite the success of penicillin in the 1940s, the major early advances in the treatment of infection occurred not through antibiosis but through chemotherapy. The principle behind chemotherapy was that there was a relationship between chemical structure and pharmacological action. The founder of this concept was Paul Erhlich (1854–1915). An early success came in 1905 when atoxyl (an organic arsenic compound) was shown to destroy trypanosomes, the microbes that caused sleeping sickness. Unfortunately, atoxyl also damaged the optic nerve. Subsequently, Erhlich and his co-workers synthesized and tested hundreds of related arsenic compounds. Ehrlich was a co-recipient (with Ilya Ilyich Mechnikov) of the Nobel Prize in medicine in 1908 for his work on immunity. Success in discovering a range of effective antibacterial drugs had three important consequences: it brought a range of important diseases under control for the first time; it provided a tremendous stimulus to research workers and opened up new avenues of research; and in the resulting commercial optimism, it led to heavy postwar investment in the pharmaceutical industry. The therapeutic revolution had begun.

3. Artificial Insemination and in Vitro Fertilization

Artificial insemination (AI) involves the extraction and collection of semen together with techniques for depositing semen in the uterus in order to achieve successful fertilization and pregnancy. Throughout the twentieth century, the approach has offered animal breeders the advantage of being able to utilize the best available breeding stock and at the correct time within the female reproductive cycle, but without the limitations of having the animals in the same location. AI has been applied most intensively within the dairy and beef cattle industries and to a lesser extent horse breeding and numerous other domesticated species.

Many of the techniques involved in artificial insemination would lay the foundation for in vitro fertilization (IVF) in the latter half of the twentieth century. IVF refers to the group of technologies that allow fertilization to take place outside the body involving the retrieval of ova or eggs from the female and sperm from the male, which are then combined in artificial, or ‘‘test tube,’’ conditions leading to fertilization. The fertilized eggs then continue to develop for several days ‘‘in culture’’ until being transferred to the female recipient to continue developing within the uterus.

4. Biopolymers

Biopolymers are natural polymers, long-chained molecules (macromolecules) consisting mostly of a repeated composition of building blocks or monomers that are formed and utilized by living organisms. Each group of biopolymers is composed of different building blocks, for example chains of sugar molecules form starch (a polysaccharide), chains of amino acids form proteins and peptides, and chains of nucleic acid form DNA and RNA (polynucleotides). Biopolymers can form gels, fibers, coatings, and films depending on the specific polymer, and serve a variety of critical functions for cells and organisms. Proteins including collagens, keratins, silks, tubulins, and actin usually form structural composites or scaffolding, or protective materials in biological systems (e.g., spider silk). Polysaccharides function in molecular recognition at cell membrane surfaces, form capsular barrier layers around cells, act as emulsifiers and adhesives, and serve as skeletal or architectural materials in plants. In many cases these polymers occur in combination with proteins to form novel composite structures such as invertebrate exoskeletons or microbial cell walls, or with lignin in the case of plant cell walls.

The use of the word ‘‘cloning’’ is fraught with confusion and inconsistency, and it is important at the outset of this discussion to offer definitional clarification. For instance, in the 1997 article by Ian Wilmut and colleagues announcing the birth of the first cloned adult vertebrate (a ewe, Dolly the sheep) from somatic cell nuclear transfer, the word clone or cloning was never used, and yet the announcement raised considerable disquiet about the prospect of cloned human beings. In a desire to avoid potentially negative forms of language, many prefer to substitute ‘‘cell expansion techniques’’ or ‘‘therapeutic cloning’’ for cloning. Cloning has been known for centuries as a horticultural propagation method: for example, plants multiplied by grafting, budding, or cuttings do not differ genetically from the original plant. The term clone entered more common usage as a result of a speech in 1963 by J.B.S. Haldane based on his paper, ‘‘Biological possibilities for the human species of the next ten-thousand years.’’ Notwithstanding these notes of caution, we can refer to a number of processes as cloning. At the close of the twentieth century, such techniques had not yet progressed to the ability to bring a cloned human to full development; however, the ability to clone cells from an adult human has potential to treat diseases. International policymaking in the late 1990s sought to distinguish between the different end uses for somatic cell nuclear transfer resulting in the widespread adoption of the distinction between ‘‘reproductive’’ and ‘‘therapeutic’’ cloning. The function of the distinction has been to permit the use (in some countries) of the technique to generate potentially beneficial therapeutic applications from embryonic stem cell technology whilst prohibiting its use in human reproduction. In therapeutic applications, nuclear transfer from a patient’s cells into an enucleated ovum is used to create genetically identical embryos that would be grown in vitro but not be allowed to continue developing to become a human being. The resulting cloned embryos could be used as a source from which to produce stem cells that can then be induced to specialize into the specific type of tissue required by the patient (such as skin for burns victims, brain neuron cells for Parkinson’s disease sufferers, or pancreatic cells for diabetics). The rationale is that because the original nuclear material is derived from a patient’s adult tissue, the risks of rejection of such cells by the immune system are reduced.

6. Gene Therapy

In 1971, Australian Nobel laureate Sir F. MacFarlane Burnet thought that gene therapy (introducing genes into body tissue, usually to treat an inherited genetic disorder) looked more and more like a case of the emperor’s new clothes. Ethical issues aside, he believed that practical considerations forestalled possibilities for any beneficial gene strategy, then or probably ever. Bluntly, he wrote: ‘‘little further advance can be expected from laboratory science in the handling of ‘intrinsic’ types of disability and disease.’’ Joshua Lederberg and Edward Tatum, 1958 Nobel laureates, theorized in the 1960s that genes might be altered or replaced using viral vectors to treat human diseases. Stanfield Rogers, working from the Oak Ridge National Laboratory in 1970, had tried but failed to cure argininemia (a genetic disorder of the urea cycle that causes neurological damage in the form of mental retardation, seizures, and eventually death) in two German girls using Swope papilloma virus. Martin Cline at the University of California in Los Angeles, made the second failed attempt a decade later. He tried to correct the bone marrow cells of two beta-thalassemia patients, one in Israel and the other in Italy. What Cline’s failure revealed, however, was that many researchers who condemned his trial as unethical were by then working toward similar goals and targeting different diseases with various delivery methods. While Burnet’s pessimism finally proved to be wrong, progress in gene therapy was much slower than antibiotic or anticancer chemotherapy developments over the same period of time. While gene therapy had limited success, it nevertheless remained an active area for research, particularly because the Human Genome Project, begun in 1990, had resulted in a ‘‘rough draft’’ of all human genes by 2001, and was completed in 2003. Gene mapping created the means for analyzing the expression patterns of hundreds of genes involved in biological pathways and for identifying single nucleotide polymorphisms (SNPs) that have diagnostic and therapeutic potential for treating specific diseases in individuals. In the future, gene therapies may prove effective at protecting patients from adverse drug reactions or changing the biochemical nature of a person’s disease. They may also target blood vessel formation in order to prevent heart disease or blindness due to macular degeneration or diabetic retinopathy. One of the oldest ideas for use of gene therapy is to produce anticancer vaccines. One method involves inserting a granulocyte-macrophage colony-stimulating factor gene into prostate tumor cells removed in surgery. The cells then are irradiated to prevent any further cancer and injected back into the same patient to initiate an immune response against any remaining metastases. Whether or not such developments become a major treatment modality, no one now believes, as MacFarland Burnet did in 1970, that gene therapy science has reached an end in its potential to advance health.

7. Genetic Engineering

The term ‘‘genetic engineering’’ describes molecular biology techniques that allow geneticists to analyze and manipulate deoxyribonucleic acid (DNA). At the close of the twentieth century, genetic engineering promised to revolutionize many industries, including microbial biotechnology, agriculture, and medicine. It also sparked controversy over potential health and ecological hazards due to the unprecedented ability to bypass traditional biological reproduction.

For centuries, if not millennia, techniques have been employed to alter the genetic characteristics of animals and plants to enhance specifically desired traits. In a great many cases, breeds with which we are most familiar bear little resemblance to the wild varieties from which they are derived. Canine breeds, for instance, have been selectively tailored to changing esthetic tastes over many years, altering their appearance, behavior and temperament. Many of the species used in farming reflect long-term alterations to enhance meat, milk, and fleece yields. Likewise, in the case of agricultural varieties, hybridization and selective breeding have resulted in crops that are adapted to specific production conditions and regional demands. Genetic engineering differs from these traditional methods of plant and animal breeding in some very important respects. First, genes from one organism can be extracted and recombined with those of another (using recombinant DNA, or rDNA, technology) without either organism having to be of the same species. Second, removing the requirement for species reproductive compatibility, new genetic combinations can be produced in a much more highly accelerated way than before. Since the development of the first rDNA organism by Stanley Cohen and Herbert Boyer in 1973, a number of techniques have been found to produce highly novel products derived from transgenic plants and animals.

At the same time, there has been an ongoing and ferocious political debate over the environmental and health risks to humans of genetically altered species. The rise of genetic engineering may be characterized by developments during the last three decades of the twentieth century.

8. Genetic Screening and Testing

The menu of genetic screening and testing technologies now available in most developed countries increased rapidly in the closing years of the twentieth century. These technologies emerged within the context of rapidly changing social and legal contexts with regard to the medicalization of pregnancy and birth and the legalization of abortion. The earliest genetic screening tests detected inborn errors of metabolism and sex-linked disorders. Technological innovations in genomic mapping and DNA sequencing, together with an explosion in research on the genetic basis of disease which culminated in the Human Genome Project (HGP), led to a range of genetic screening and testing for diseases traditionally recognized as genetic in origin and for susceptibility to more common diseases such as certain types of familial cancer, cardiac conditions, and neurological disorders among others. Tests were also useful for forensic, or nonmedical, purposes. Genetic screening techniques are now available in conjunction with in vitro fertilization and other types of reproductive technologies, allowing the screening of fertilized embryos for certain genetic mutations before selection for implantation. At present selection is purely on disease grounds and selection for other traits (e.g., for eye or hair color, intelligence, height) cannot yet be done, though there are concerns for eugenics and ‘‘designer babies.’’ Screening is available for an increasing number of metabolic diseases through tandem mass spectrometry, which uses less blood per test, allows testing for many conditions simultaneously, and has a very low false-positive rate as compared to conventional Guthrie testing. Finally, genetic technologies are being used in the judicial domain for determination of paternity, often associated with child support claims, and for forensic purposes in cases where DNA material is available for testing.

9. Plant Breeding: Genetic Methods

The cultivation of plants is the world’s oldest biotechnology. We have continually tried to produce improved varieties while increasing yield, features to aid cultivation and harvesting, disease, and pest resistance, or crop qualities such as longer postharvest storage life and improved taste or nutritional value. Early changes resulted from random crosspollination, rudimentary grafting, or spontaneous genetic change. For centuries, man kept the seed from the plants with improved characteristics to plant the following season’s crop. The pioneering work of Gregor Mendel and his development of the basic laws of heredity showed for other first time that some of the processes of heredity could be altered by experimental means. The genetic analysis of bacterial (prokaryote) genes and techniques for analysis of the higher (eukaryotic) organisms such as plants developed in parallel streams, but the rediscovery of Mendel’s work in 1900 fueled a burst of activity on understanding the role of genes in inheritance. The knowledge that genes are linked along the chromosome thereby allowed mapping of genes (transduction analysis, conjugation analysis, and transformation analysis). The power of genetics to produce a desirable plant was established, and it was appreciated that controlled breeding (test crosses and back crosses) and careful analysis of the progeny could distinguish traits that were dominant or recessive, and establish pure breeding lines. Traditional horticultural techniques of artificial self-pollination and cross-pollination were also used to produce hybrids. In the 1930s the Russian Nikolai Vavilov recognized the value of genetic diversity in domesticated crop plants and their wild relatives to crop improvement, and collected seeds from the wild to study total genetic diversity and use these in breeding programs. The impact of scientific crop breeding was established by the ‘‘Green revolution’’ of the 1960s, when new wheat varieties with higher yields were developed by careful crop breeding. ‘‘Mutation breeding’’— inducing mutations by exposing seeds to x-rays or chemicals such as sodium azide, accelerated after World War II. It was also discovered that plant cells and tissues grown in tissue culture would mutate rapidly. In the 1970s, haploid breeding, which involves producing plants from two identical sets of chromosomes, was extensively used to create new cultivars. In the twenty-first century, haploid breeding could speed up plant breeding by shortening the breeding cycle.

10. Tissue Culturing

The technique of tissue or cell culture, which relates to the growth of tissue or cells within a laboratory setting, underlies a phenomenal proportion of biomedical research. Though it has roots in the late nineteenth century, when numerous scientists tried to grow samples in alien environments, cell culture is credited as truly beginning with the first concrete evidence of successful growth in vitro, demonstrated by Johns Hopkins University embryologist Ross Harrison in 1907. Harrison took sections of spinal cord from a frog embryo, placed them on a glass cover slip and bathed the tissue in a nutrient media. The results of the experiment were startling—for the first time scientists visualized actual nerve growth as it would happen in a living organism—and many other scientists across the U.S. and Europe took up culture techniques. Rather unwittingly, for he was merely trying to settle a professional dispute regarding the origin of nerve fibers, Harrison fashioned a research tool that has since been designated by many as the greatest advance in medical science since the invention of the microscope.

From the 1980s, cell culture has once again been brought to the forefront of cancer research in the isolation and identification of numerous cancer causing oncogenes. In addition, cell culturing continues to play a crucial role in fields such as cytology, embryology, radiology, and molecular genetics. In the future, its relevance to direct clinical treatment might be further increased by the growth in culture of stem cells and tissue replacement therapies that can be tailored for a particular individual. Indeed, as cell culture approaches its centenary, it appears that its importance to scientific, medical, and commercial research the world over will only increase in the twenty-first century.

History of Biotechnology

Biotechnology grew out of the technology of fermentation, which was called zymotechnology. This was different from the ancient craft of brewing because of its thought-out relationships to science. These were most famously conceptualized by the Prussian chemist Georg Ernst Stahl (1659–1734) in his 1697 treatise Zymotechnia Fundamentalis, in which he introduced the term zymotechnology. Carl Balling, long-serving professor in Prague, the world center of brewing, drew on the work of Stahl when he published his Bericht uber die Fortschritte der zymotechnische Wissenschaften und Gewerbe (Account of the Progress of the Zymotechnic Sciences and Arts) in the mid-nineteenth century. He used the idea of zymotechnics to compete with his German contemporary Justus Liebig for whom chemistry was the underpinning of all processes.

By the end of the nineteenth century, there were attempts to develop a new scientific study of fermentation. It was an aspect of the ‘‘second’’ Industrial Revolution during the period from 1870 to 1914. The emergence of the chemical industry is widely taken as emblematic of the formal research and development taking place at the time. The development of microbiological industries is another example. For the first time, Louis Pasteur’s germ theory made it possible to provide convincing explanations of brewing and other fermentation processes.

Pasteur had published on brewing in the wake of France’s humiliation in the Franco–Prussian war (1870–1871) to assert his country’s superiority in an industry traditionally associated with Germany. Yet the science and technology of fermentation had a wide range of applications including the manufacture of foods (cheese, yogurt, wine, vinegar, and tea), of commodities (tobacco and leather), and of chemicals (lactic acid, citric acid, and the enzyme takaminase). The concept of zymotechnology associated principally with the brewing of beer began to appear too limited to its principal exponents. At the time, Denmark was the world leader in creating high-value agricultural produce. Cooperative farms pioneered intensive pig fattening as well as the mass production of bacon, butter, and beer. It was here that the systems of science and technology were integrated and reintegrated, conceptualized and reconceptualized.

The Dane Emil Christian Hansen discovered that infection from wild yeasts was responsible for numerous failed brews. His contemporary Alfred Jørgensen, a Copenhagen consultant closely associated with the Tuborg brewery, published a widely used textbook on zymotechnology. Microorganisms and Fermentation first appeared in Danish 1889 and would be translated, reedited, and reissued for the next 60 years.

The scarcity of resources on both sides during World War I brought together science and technology, further development of zymotechnology, and formulation of the concept of biotechnology. Impending and then actual war accelerated the use of fermentation technologies to make strategic materials. In Britain a variant of a process to ferment starch to make butadiene for synthetic rubber production was adapted to make acetone needed in the manufacture of explosives. The process was technically important as the first industrial sterile fermentation and was strategically important for munitions supplies. The developer, chemist Chaim Weizmann, later became well known as the first president of Israel in 1949.

In Germany scarce oil-based lubricants were replaced by glycerol made by fermentation. Animal feed was derived from yeast grown with the aid of the new synthetic ammonia in another wartime development that inspired the coining of the word biotechnology. Hungary was the agricultural base of the Austro–Hungarian empire and aspired to Danish levels of efficiency. The economist Karl Ereky (1878–1952) planned to go further and build the largest industrial pig-processing factory. He envisioned a site that would fatten 50,000 swine at a time while railroad cars of sugar beet arrived and fat, hides, and meat departed. In this forerunner of the Soviet collective farm, peasants (in any case now falling prey to the temptations of urban society) would be completely superseded by the industrialization of the biological process in large factory-like animal processing units. Ereky went further in his ruminations over the meaning of his innovation. He suggested that it presaged an industrial revolution that would follow the transformation of chemical technology. In his book entitled Biotechnologie, he linked specific technical injunctions to wide-ranging philosophy. Ereky was neither isolated nor obscure. He had been trained in the mainstream of reflection on the meaning of the applied sciences in Hungary, which would be remarkably productive across the sciences. After World War I, Ereky served as Hungary’s minister of food in the short-lived right wing regime that succeeded the fall of the communist government of Bela Kun.

Nonetheless it was not through Ereky’s direct action that his ideas seem to have spread. Rather, his book was reviewed by the influential Paul Lindner, head of botany at the Institut fu¨ r Ga¨ rungsgewerbe in Berlin, who suggested that microorganisms could also be seen as biotechnological machines. This concept was already found in the production of yeast and in Weizmann’s work with strategic materials, which was widely publicized at that very time. It was with this meaning that the word ‘‘Biotechnologie’’ entered German dictionaries in the 1920s.

Biotechnology represented more than the manipulation of existing organisms. From the beginning it was concerned with their improvement as well, and this meant the enhancement of all living creatures. Most dramatically this would include humanity itself; more mundanely it would include plants and animals of agricultural importance. The enhancement of people was called eugenics by the Victorian polymath and cousin of Charles Darwin, Francis Galton. Two strains of eugenics emerged: negative eugenics associated with weeding out the weak and positive eugenics associated with enhancing strength. In the early twentieth century, many eugenics proponents believed that the weak could be made strong. People had after all progressed beyond their biological limits by means of technology.

Jean-Jacques Virey, a follower of the French naturalist Jean-Baptiste de Monet de Lamarck, had coined the term ‘‘biotechnie’’ in 1828 to describe man’s ability to make technology do the work of biology, but it was not till a century later that the term entered widespread use. The Scottish biologist and town planner Patrick Geddes made biotechnics popular in the English-speaking world. Geddes, too, sought to link life and technology. Before World War I he had characterized the technological evolution of mankind as a move from the paleotechnic era of coal and iron to the neotechnic era of chemicals, electricity, and steel. After the war, he detected a new era based on biology—the biotechnic era. Through his friend, writer Lewis Mumford, Geddes would have great influence. Mumford’s book Technics and Civilization, itself a founding volume of the modern historiography of technology, promoted his vision of the Geddesian evolution.

A younger generation of English experimental biologists with a special interest in genetics, including J. B. S. Haldane, Julian Huxley, and Lancelot Hogben, also promoted a concept of biotechnology in the period between the world wars. Because they wrote popular works, they were among Britain’s best-known scientists. Haldane wrote about biological invention in his far-seeing work Daedalus. Huxley looked forward to a blend of social and eugenics-based biological engineering. Hogben, following Geddes, was more interested in engineering plants through breeding. He tied the progressivism of biology to the advance of socialism.

The improvement of the human race, genetic manipulation of bacteria, and the development of fermentation technology were brought together by the development of penicillin during World War II. This drug was successfully extracted from the juice exuded by a strain of the Penicillium fungus. Although discovered by accident and then developed further for purely scientific reasons, the scarce and unstable ‘‘antibiotic’’ called penicillin was transformed during World War II into a powerful and widely used drug. Large networks of academic and government laboratories and pharmaceutical manufacturers in Britain and the U.S. were coordinated by agencies of the two governments. An unanticipated combination of genetics, biochemistry, chemistry, and chemical engineering skills had been required. When the natural mold was bombarded with high-frequency radiation, far more productive mutants were produced, and subsequently all the medicine was made using the product of these man-made cells. By the 1950s penicillin was cheap to produce and globally available.

The new technology of cultivating and processing large quantities of microorganisms led to calls for a new scientific discipline. Biochemical engineering was one term, and applied microbiology another. The Swedish biologist, Carl-Goran Heden, possibly influenced by German precedents, favored the term ‘‘Biotechnologi’’ and persuaded his friend Elmer Gaden to relabel his new journal Biotechnology and Biochemical Engineering. From 1962 major international conferences were held under the banner of the Global Impact of Applied Microbiology. During the 1960s food based on single-cell protein grown in fermenters on oil or glucose seemed, to visionary engineers and microbiologists and to major companies, to offer an immediate solution to world hunger. Tropical countries rich in biomass that could be used as raw material for fermentation were also the world’s poorest. Alcohol could be manufactured by fermenting such starch or sugar rich crops as sugar cane and corn. Brazil introduced a national program of replacing oil-based petrol with alcohol in the 1970s.

It was not, however, just the developing countries that hoped to benefit. The Soviet Union developed fermentation-based protein as a major source of animal feed through the 1980s. In the U.S. it seemed that oil from surplus corn would solve the problem of low farm prices aggravated by the country’s boycott of the USSR in1979, and the term ‘‘gasohol‘‘ came into currency. Above all, the decline of established industries made the discovery of a new wealth maker an urgent priority for Western governments. Policy makers in both Germany and Japan during the 1970s were driven by a sense of the inadequacy of the last generation of technologies. These were apparently maturing, and the succession was far from clear. Even if electronics or space travel offered routes to the bright industrial future, these fields seemed to be dominated by the U.S. Seeing incipient crisis, the Green, or environmental, movement promoted a technology that would depend on renewable resources and on low-energy processes that would produce biodegradable products, recycle waste, and address problems of the health and nutrition of the world.

In 1973 the German government, seeking a new and ‘‘greener’’ industrial policy, commissioned a report entitled Biotechnologie that identified ways in which biological processing was key to modern developments in technology. Even though the report was published at the time that recombinant DNA (deoxyribonucleic acid) was becoming possible, it did not refer to this new technique and instead focused on the use and combination of existing technologies to make novel products.

Nonetheless the hitherto esoteric science of molecular biology was making considerable progress, although its practice in the early 1970s was rather distant from the world of industrial production. The phrase ‘‘genetic engineering’’ entered common parlance in the 1960s to describe human genetic modification. Medicine, however, put a premium on the use of proteins that were difficult to extract from people: insulin for diabetics and interferon for cancer sufferers. During the early 1970s what had been science fiction became fact as the use of DNA synthesis, restriction enzymes, and plasmids were integrated. In 1973 Stanley Cohen and Herbert Boyer successfully transferred a section of DNA from one E. coli bacterium to another. A few prophets such as Joshua Lederberg and Walter Gilbert argued that the new biological techniques of recombinant DNA might be ideal for making synthetic versions of expensive proteins such as insulin and interferon through their expression in bacterial cells. Small companies, such as Cetus and Genentech in California and Biogen in Cambridge, Massachusetts, were established to develop the techniques. In many cases discoveries made by small ‘‘boutique’’ companies were developed for the market by large, more established, pharmaceutical organizations.

Many governments were impressed by these advances in molecular genetics, which seemed to make biotechnology a potential counterpart to information technology in a third industrial revolution. These inspired hopes of industrial production of proteins identical to those produced in the human body that could be used to treat genetic diseases. There was also hope that industrially useful materials such as alcohol, plastics (biopolymers), or ready-colored fibers might be made in plants, and thus the attractions of a potentially new agricultural era might be as great as the implications for medicine. At a time of concern over low agricultural prices, such hopes were doubly welcome. Indeed, the agricultural benefits sometimes overshadowed the medical implications.

The mechanism for the transfer of enthusiasm from engineering fermenters to engineering genes was the New York Stock Exchange. At the end of the 1970s, new tax laws encouraged already adventurous U.S. investors to put money into small companies whose stock value might grow faster than their profits. The brokerage firm E. F. Hutton saw the potential for the new molecular biology companies such as Biogen and Cetus. Stock market interest in companies promising to make new biological entities was spurred by the 1980 decision of the U.S. Supreme Court to permit the patenting of a new organism. The patent was awarded to the Exxon researcher Ananda Chakrabarty for an organism that metabolized hydrocarbon waste. This event signaled the commercial potential of biotechnology to business and governments around the world. By the early 1980s there were widespread hopes that the protein interferon, made with some novel organism, would provide a cure for cancer. The development of monoclonal antibody technology that grew out of the work of Georges J. F. Kohler and Cesar Milstein in Cambridge (co-recipients with Niels K. Jerne of the Nobel Prize in medicine in 1986) seemed to offer new prospects for precise attacks on particular cells.

The fear of excessive regulatory controls encouraged business and scientific leaders to express optimistic projections about the potential of biotechnology. The early days of biotechnology were fired by hopes of medical products and high-value pharmaceuticals. Human insulin and interferon were early products, and a second generation included the anti-blood clotting agent tPA and the antianemia drug erythropoietin. Biotechnology was also used to help identify potential new drugs that might be made chemically, or synthetically.

At the same time agricultural products were also being developed. Three early products that each raised substantial problems were bacteria which inhibited the formation of frost on the leaves of strawberry plants (ice-minus bacteria), genetically modified plants including tomatoes and rapeseed, and the hormone bovine somatrotropin (BST) produced in genetically modified bacteria and administered to cattle in the U.S. to increase milk yields. By 1999 half the soy beans and one third of the corn grown in the U.S. were modified. Although the global spread of such products would arouse the best known concern at the end of the century, the use of the ice-minus bacteria— the first authorized release of a genetically engineered organism into the environment—had previously raised anxiety in the U.S. in the 1980s.

In 1997 Dolly the sheep was cloned from an adult mother in the Roslin agricultural research institute outside Edinburgh, Scotland. This work was inspired by the need to find a way of reproducing sheep engineered to express human proteins in their milk. However, the public interest was not so much in the cloning of sheep that had just been achieved as in the cloning of people, which had not. As in the Middle Ages when deformed creatures had been seen as monsters and portents of natural disasters, Dolly was similarly seen as monster and as a portent of human cloning.

The name Frankenstein, recalled from the story written by Mary Shelley at the beginning of the nineteenth century and from the movies of the 1930s, was once again familiar at the end of the twentieth century. Shelley had written in the shadow of Stahl’s theories. The continued appeal of this book embodies the continuity of the fears of artificial life and the anxiety over hubris. To this has been linked a more mundane suspicion of the blending of commerce and the exploitation of life. Discussion of biotechnology at the end of the twentieth century was therefore colored by questions of whose assurances of good intent and reassurance of safety could be trusted.

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Top 50 Emerging Research Topics in Biotechnology

Trending Research Topics in Biotechnology

Biotechnology is a dynamic field that continuously shapes our world, enabling innovation, breakthroughs, and solutions to various challenges. As we move into the future, numerous emerging research areas promise to revolutionize healthcare, agriculture, environmental sustainability, and more. The top 50 emerging research topics in biotechnology are presented in this article.

1. Gene Editing and Genomic Engineering

a. CRISPR and Gene Editing

Precision Medicine : Developing targeted therapies for various diseases using CRISPR/Cas9 and other gene-editing tools.

Ethical Implications : Exploring and addressing ethical concerns surrounding CRISPR use in human embryos and germline editing.

Agricultural Advancements : Enhancing crop resistance and nutritional content through gene editing of improved farm outcomes.

Gene Drive Technology : Investigating the potential of gene drive technology to control vector-borne diseases like malaria and dengue fever.

Regulatory Frameworks : Establishing global regulations for responsible gene editing applications in different fields.

b. Synthetic Biology

Bioengineering Microbes : Creating engineered microorganisms for sustainable production of fuels, pharmaceuticals, and materials.

Designer Organisms : Designing novel organisms with specific functionalities for environmental remediation or industrial processes.

Cell-Free Systems : Developing cell-free systems for various applications, including drug production and biosensors.

Biosecurity Measures : Addressing concerns regarding the potential misuse of synthetic biology for bioterrorism.

Standardization and Automation : Standardizing synthetic biology methodologies and automating processes to streamline production.

2. Personalized Medicine and Pharmacogenomics

a. Precision Medicine

Individualized Treatment : Tailoring medical treatment based on a person’s genetic makeup and environmental factors.

Cancer Therapy : Advancing targeted cancer therapies based on the genetic profile of tumors and patients.

Data Analytics : Implementing big data and AI for comprehensive analysis of genomic and clinical data to improve treatment outcomes.

Clinical Implementation : Integrating genetic testing into routine clinical practice for personalized healthcare.

Public Health and Policy : Addressing the challenges of integrating personalized medicine into public health policies and practices.

b. Pharmacogenomics

Drug Development : Optimizing drug development based on individual genetic variations to improve efficacy and reduce side effects.

Adverse Drug Reactions : Understanding genetic predispositions to adverse drug reactions and minimizing risks.

Dosing Optimization : Tailoring drug dosage based on an individual’s genetic profile for better treatment outcomes.

Economic Implications : Assessing the economic impact of pharmacogenomics on healthcare systems.

Education and Training : Educating healthcare professionals on integrating pharmacogenomic data into clinical practice.

3. Nanobiotechnology and Nanomedicine

a. Nanoparticles in Medicine

Drug Delivery Systems : Developing targeted drug delivery systems using nanoparticles for enhanced efficacy and reduced side effects.

Theranostics : Integrating diagnostics and therapeutics through nanomaterials for personalized medicine.

Imaging Techniques : Advancing imaging technologies using nanoparticles for better resolution and early disease detection.

Biocompatibility and Safety : Ensuring the safety and biocompatibility of nanoparticles used in medicine.

Regulatory Frameworks : Establishing regulations for the use of nanomaterials in medical applications.

b. Nanosensors and Diagnostics

Point-of-Care Diagnostics : Developing portable and rapid diagnostic tools for various diseases using nanotechnology.

Biosensors : Creating highly sensitive biosensors for detecting biomarkers and pathogens in healthcare and environmental monitoring.

Wearable Health Monitors : Integrating nanosensors into wearable devices for continuous health monitoring.

Challenges and Limitations : Addressing challenges in scalability, reproducibility, and cost-effectiveness of nanosensor technologies.

Future Applications : Exploring potential applications of nanosensors beyond healthcare, such as environmental monitoring and food safety.

4. Immunotherapy and Vaccine Development

a. Cancer Immunotherapy

Immune Checkpoint Inhibitors : Enhancing the efficacy of immune checkpoint inhibitors and understanding resistance mechanisms.

CAR-T Cell Therapy : Improving CAR-T cell therapy for a wider range of cancers and reducing associated side effects.

Combination Therapies : Investigating combination therapies for better outcomes in cancer treatment.

Biomarkers and Predictive Models : Identifying predictive biomarkers for immunotherapy response.

Long-Term Effects : Studying the long-term effects and immune-related adverse events of immunotherapies.

b. Vaccine Technology

mRNA Vaccines : Advancing mRNA vaccine technology for various infectious diseases and cancers.

Universal Vaccines : Developing universal vaccines targeting multiple strains of viruses and bacteria.

Vaccine Delivery Systems : Innovating vaccine delivery methods for improved stability and efficacy.

Vaccine Hesitancy : Addressing vaccine hesitancy through education, communication, and community engagement.

Pandemic Preparedness : Developing strategies for rapid vaccine development and deployment during global health crises.

5. Environmental Biotechnology and Sustainability

a. Bioremediation and Bioenergy

Biodegradation Techniques : Using biotechnology to enhance the degradation of pollutants and contaminants in the environment.

Biofuels : Developing sustainable biofuel production methods from renewable resources.

Microbial Fuel Cells : Harnessing microbial fuel cells for energy generation from organic waste.

Circular Economy : Integrating biotechnological solutions for a circular economy and waste management.

Ecosystem Restoration : Using biotechnology for the restoration of ecosystems affected by pollution and climate change.

b. Agricultural Biotechnology

Genetically Modified Crops : Advancing genetically modified crops for improved yields, pest resistance, and nutritional content.

Precision Agriculture : Implementing biotechnological tools for precise and sustainable farming practices.

Climate-Resilient Crops : Developing crops resilient to climate change-induced stresses.

Micro-biome Applications : Leveraging the plant micro-biome for enhanced crop health and productivity.

Consumer Acceptance and Regulation : Addressing consumer concerns and regulatory challenges related to genetically modified crops.

The field of biotechnology is a beacon of hope for addressing the challenges of our time, offering promising solutions for healthcare, sustainability, and more. As researchers explore these emerging topics, the potential for ground-breaking discoveries and transformative applications is immense.

I hope this article will help you to find the top research topics in biotechnology that promise to revolutionize healthcare, agriculture, environmental sustainability, and more.

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Biotechnology

Research in biotechnology can helps in bringing massive changes in humankind and lead to a better life. In the last few years, there have been so many leaps, and paces of innovations as scientists worldwide worked to develop and produce novel mRNA vaccinations and brought some significant developments in biotechnology. During this period, they also faced many challenges. Disturbances in the supply chain and the pandemic significantly impacted biotech labs and researchers, forcing lab managers to become ingenious in buying lab supplies, planning experiments, and using technology for maintaining research schedules.

The Biotech Research Technique is changing

How research is being done is changing, as also how scientists are conducting it. Affected by both B2C eCommerce and growing independence in remote and cloud-dependent working, most of the biotechnology labs are going through some digital transformations. This implies more software, automation, and AI in the biotech lab, along with some latest digital procurement plans and integrated systems for various lab operations.

Look at some of the top trends in biotech research and recent Biotechnology Topics that are bringing massive changes in this vast world of science, resulting in some innovation in life sciences and biotechnology ideas .

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Current research in biotechnology: Exploring the biotech forefront

2019, Current Research in Biotechnology

Biotechnology is an evolving research field that covers a broad range of topics. Here we aimed to evaluate the latest research literature, to identify prominent research themes, major contributors in terms of institutions, countries/re-gions, and journals. The Web of Science Core Collection online database was searched to retrieve biotechnology articles published since 2017. In total, 12,351 publications were identified and analyzed. Over 8500 institutions contributed to these biotechnology publications, with the top 5 most productive ones scattered over France, China, the United States of America, Spain, and Brazil. Over 140 countries/regions contributed to the biotechnology research literature, led by the United States of America, China, Germany, Brazil, and India. Journal of Bioscience and Bioengineer-ing was the most productive journal in terms of number of publications. Metabolic engineering was among the most prevalent biotechnology study themes, and Escherichia coli and Saccharomyces cerevisiae were frequently used in biotechnology investigations, including the biosynthesis of useful biomolecules, such as myo-inositol (vitamin B8), mono-terpenes, adipic acid, astaxanthin, and ethanol. Nanoparticles and nanotechnology were identified too as emerging biotechnology research themes of great significance. Biotechnology continues to evolve and will remain a major driver of societal innovation and development.

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Scientists worldwide are continuing to discover unique properties of every day materials at the submicrometer scale. This size domain is better known as nanometer domain and technology concerned with this is known as nanotechnology that involves working with particles at nano level. One of the most important emerging fields of science in this centur y is Nanotechnology. It deals with designing, construction, investigation and utilization of systems at the nanoscale. The interface between nanotechnology and biotechnology is nanobiotechnology, which exploits nanotechnology and biotechnology to analyse a nd create nanobiosystems to meet a wide variety of challenges and develops a wide range of applications. Biotechnology gives us a way to understand biological system and to utilize our knowledge for industrial manufacturing. Nanotechnology has great potent ial and by the help of its application it can enhance the quality of life through in various fields like agriculture and the food system. Around the world, it has become the future of any nation. Important tools used in nanotechnology and application of nanobiotechnology in agriculture sector will be discussed in this review.

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An Introduction to Biotechnology

Varsha gupta.

5 Institute of Biosciences and Biotechnology, Chhatrapati Shahu Ji Maharaj University, Kanpur, UP India

Manjistha Sengupta

6 George Washington University, Washington DC, USA

Jaya Prakash

7 Orthopaedics Unit, Community Health Centre, Kanpur, UP India

Baishnab Charan Tripathy

8 School of Life sciences, Jawaharlal Nehru University, New Delhi, India

Biotechnology is multidisciplinary field which has major impact on our lives. The technology is known since years which involves working with cells or cell-derived molecules for various applications. It has wide range of uses and is termed “technology of hope” which impact human health, well being of other life forms and our environment. It has revolutionized diagnostics and therapeutics; however, the major challenges to the human beings have been threats posed by deadly virus infections as avian flu, Chikungunya, Ebola, Influenza A, SARS, West Nile, and the latest Zika virus. Personalized medicine is increasingly recognized in healthcare system. In this chapter, the readers would understand the applications of biotechnology in human health care system. It has also impacted the environment which is loaded by toxic compounds due to human industrialization and urbanization. Bioremediation process utilizes use of natural or recombinant organisms for the cleanup of environmental toxic pollutants. The development of insect and pest resistant crops and herbicide tolerant crops has greatly reduced the environmental load of toxic insecticides and pesticides. The increase in crop productivity for solving world food and feed problem is addressed in agricultural biotechnology. The technological advancements have focused on development of alternate, renewable, and sustainable energy sources for production of biofuels. Marine biotechnology explores the products which can be obtained from aquatic organisms. As with every research area, the field of biotechnology is associated with many ethical issues and unseen fears. These are important in defining laws governing the feasibility and approval for the conduct of particular research.

Introduction

The term “ biotechnology” was coined by a Hungarian engineer Karl Ereky, in 1919, to refer to the science and methods that permit products to be produced from raw materials with the aid of living organisms. Biotechnology is a diverse field which involves either working with living cells or using molecules derived from them for applications oriented toward human welfare using varied types of tools and technologies. It is an amalgamation of biological science with engineering whereby living organisms or cells or parts are used for production of products and services. The main subfields of biotechnology are medical (red) biotechnology, agricultural (green) biotechnology, industrial (white) biotechnology, marine (blue) biotechnology, food biotechnology, and environmental biotechnology (Fig. 1.1 .). In this chapter the readers will understand the potential applications of biotechnology in several fields like production of medicines; diagnostics; therapeutics like monoclonal antibodies, stem cells, and gene therapy; agricultural biotechnology; pollution control ( bioremediation); industrial and marine biotechnology; and biomaterials, as well as the ethical and safety issues associated with some of the products.

Major applications of biotechnology in different areas and some of their important products

The biotechnology came into being centuries ago when plants and animals began to be selectively bred and microorganisms were used to make beer, wine, cheese, and bread. However, the field gradually evolved, and presently it is the use or manipulation of living organisms to produce beneficiary substances which may have medical, agricultural, and/or industrial utilization. Conventional biotechnology is referred to as the technique that makes use of living organism for specific purposes as bread/cheese making, whereas modern biotechnology deals with the technique that makes use of cellular molecules like DNA, monoclonal antibodies, biologics, etc. Before we go into technical advances of DNA and thus recombinant DNA technology, let us have the basic understanding about DNA and its function.

The foundation of biotechnology was laid down after the discovery of structure of DNA in the early 1950s. The hereditary material is deoxyribonucleic acid (DNA) which contains all the information that dictates each and every step of an individual’s life. The DNA consists of deoxyribose sugar, phosphate, and four nitrogenous bases (adenine, guanine, cytosine, and thymine). The base and sugar collectively form nucleoside, while base, sugar, and phosphate form nucleotide (Fig. 1.2 ). These are arranged in particular orientation on DNA called order or sequence and contain information to express them in the form of protein. DNA has double helical structure, with two strands being complimentary and antiparallel to each other, in which A on one strand base pairs with T and G base pairs with C with two and three bonds, respectively. DNA is the long but compact molecule which is nicely packaged in our nucleus. The DNA is capable of making more copies like itself with the information present in it, as order or sequence of bases. This is called DNA replication. When the cell divides into two, the DNA also replicates and divides equally into two. The process of DNA replication is shown in Fig. 1.3 , highlighting important steps.

The double helical structure of DNA where both strands are running in opposite direction. Elongation of the chain occurs due to formation of phosphodiester bond between phosphate at 5′ and hydroxyl group of sugar at 3′ of the adjacent sugar of the nucleotide in 5–3′ direction. The sugar is attached to the base. Bases are of four kinds: adenine ( A ), guanine ( G ) (purines), thymine ( T ), and cytosine ( C ) (pyrimidines). Adenine base pairs with two hydrogen bonds with thymine on the opposite antiparallel strand and guanine base pairs with three hydrogen bonds with cytosine present on the opposite antiparallel strand

The process of DNA replication. The DNA is densely packed and packaged in the chromosomes. The process requires the action of several factors and enzymes. DNA helicase unwinds the double helix. Topoisomerase relaxes DNA from its super coiled nature. Single-strand binding proteins bind to single-stranded open DNA and prevent its reannealing and maintains strand separation. DNA polymerase is an enzyme which builds a new complimentary DNA strand and has proofreading activity. DNA clamp is a protein which prevents dissociation of DNA polymerase. Primase provides a short RNA sequence for DNA polymerase to begin synthesis. DNA ligase reanneals and joins the Okazaki fragments of the lagging strand. DNA duplication follows semiconservative replication, where each strand serves as template which leads to the production of two complimentary strands. In the newly formed DNA, one strand is old and the other one is new (semiconservative replication). DNA polymerase can extend existing short DNA or RNA strand which is paired to template strand and is called primer. Primer is required as DNA polymerase cannot start the synthesis directly. DNA polymerase is capable of proofreading, that is, correction of wrongly incorporated nucleotide. One strand is replicated continuously with single primer, and it is called as leading strand. Other strand is discontinuous and requires the addition of several primers. The extension is done in the form of short fragments called as Okazaki fragments. The gaps are sealed by DNA ligase. Replication always occurs in 5′–3′ direction

DNA contains whole information for the working of the cell. The part of the DNA which has information to dictate the biosynthesis of a polypeptide is called a “gene.” The arrangement or order of nucleotides determines the kind of proteins which we produce. Each gene is responsible for coding a functional polypeptide. The genes have the information to make a complimentary copy of mRNA. The information of DNA which makes RNA in turn helps cells to incorporate amino acids according to arrangement of letters for making many kinds of proteins. These letters are transcribed into mRNA in the form of triplet codon, where each codon specifies one particular amino acid. The polypeptide is thus made by adding respective amino acids according to the instructions present on RNA. Therefore, the arrangement of four bases (adenine, guanine, cytosine, and thymine) dictates the information to add any of the 20 amino acids to make all the proteins in all the living organisms. Few genes need to be expressed continuously, as their products are required by the cell, and these are known as “constitutive genes.” They are responsible for basic housekeeping functions of the cells. However, depending upon the physiological demand and cell’s requirement at a particular time, some genes are active and some are inactive, that is, they do not code for any protein. The information contained in the DNA is used to make mRNA in the process of “ transcription” (factors shown in Table 1.1 ). The information of mRNA is used in the process of “ translation” for production of protein. Transcription occurs in the nucleus and translation in the cytoplasm of the cell. In translation several initiation factors help in the assembly of mRNA with 40S ribosome and prevent binding of both ribosomal subunits; they also associate with cap and poly(A) tail. Several elongation factors play an important role in chain elongation. Though each cell of the body has the same genetic makeup, but each is specialized to perform unique functions, the activation and expression of genes is different in each cell. Thus, one type of cells can express some of its genes at one time and may not express the same genes some other time. This is called “temporal regulation” of the gene. In the body different cells express different genes and thus different proteins. For example, liver cell and lymphocyte, would express different genes. This is known as spatial regulation of the gene. Therefore, in the cells of the body, the activation of genes is under spatial regulation (cells present at different locations and different organs produce different proteins) and temporal regulation (same cells produce different proteins at different times). The proteins are formed by the information contained in the DNA and perform a variety of cellular functions. The proteins may be structural (responsible for cell shape and size), or they may be functional like enzymes, signaling intermediates, regulatory proteins, and defense system proteins. However, any kind of genetic defect results in defective protein or alters protein folding which can compromise the functioning of the body and is responsible for the diseases. Figure 1.4 shows the outline of the process of transcription and translation with important steps.

Factors involved in transcription process

It shows the process of transcription and translation. Transcription occurs in the nucleus and requires the usage of three polymerase enzymes. RNApol I for rRNA, pol II for mRNA, and pol III for both rRNA and tRNA. RNApol II initiates the process by associating itself with seven transcription factors, TFIIA, TFIIB, TFIID, TFIIE, TFIIH, and TFIIJ. After the synthesis, preRNA transcript undergoes processing to form mRNA by removal of introns by splicing and polyadenylation and capping. Protein synthesis is initiated by formation of ribosome and initiator tRNA complex to initiation codon (AUG) of mRNA and involves 11 factors. Chain elongation occurs after sequential addition of amino acids by formation of peptide bonds. Then polypeptide can fold or conjugate itself to other biomolecules and may undergo posttranslational modifications as glycosylation or phosphorylation to perform its biological functions

The biotechnological tools are employed toward modification of the gene for gain of function or loss of function of the protein. The technique of removing, adding, or modifying genes in the genome or chromosomes of an organism to bring about the changes in the protein information is called genetic engineering or recombinant DNA technology (Fig. 1.5 ). DNA recombination made possible the sequencing of the human genome and laid the foundation for the nascent fields of bioinformatics, nanomedicine, and individualized therapy. Multicellular organisms like cows, goats, sheep, rats, corn, potato, and tobacco plants have been genetically engineered to produce substances medically useful to humans. Genetic engineering has revolutionized medicine, enabling mass production of safe, pure, more effective versions of biochemicals that the human body produces naturally [ 20 – 22 ].

The process of recombinant DNA technology. The gene of interest from human nucleus is isolated and cloned in a plasmid vector. The gene is ligated with the help of DNA ligase. The vector is transformed into a bacterial host. After appropriate selections, the gene is amplified when bacteria multiply or the gene can be sequenced or the gene can be expressed to produce protein

The technological advancements have resulted in (1) many biopharmaceuticals and vaccines, (2) new and specific ways to diagnose, (3) increasing the productivity and introduction of quality traits in agricultural crops, (4) the ways to tackle the pollutants efficiently for sustainable environmental practices, (5) helped the forensic experts by DNA fingerprinting and profiling, (6) fermentation technology for production of industrially important products. The list is very long with tremendous advancements and products which have boosted the economy of biotechnology sector worldwide [ 16 ]. The biotechnology industry and the products are regulated by various government organizations such as the US Food and Drug Administration (FDA), the Environmental Protection Agency (EPA), and the US Department of Agriculture (USDA).

Medical Biotechnology

This fieldof biotechnology has many applications and is involved in production of recombinant pharmaceuticals, tissue engineering products, regenerative medicines such as stem cell and gene therapy, and many more biotechnology products for better human life (Fig. 1.6 ). Biotechnological tools produce purified bio-therapeutic agents on industrial scales. These include both novel agents and agents formerly available only in small quantities. Crude vaccines were used in antiquity in China, India, and Persia. For example, vaccination with scabs that contained the smallpox virus was a practice in the East for centuries. In 1798 English country doctor Edward Jenner demonstrated that inoculation with pus from sores due to infection by a related cowpox virus could prevent smallpox far less dangerously. It marked the beginning of vaccination. Humans have been benefited incalculably from the implementation of vaccination programs.

Various applications of medical biotechnology

Tremendous progress has been made since the early recombinant DNA technology (RDT) experiments from which the lively—and highly profitable—biotechnology industry emerged. RDT has fomented multiple revolutions in medicine. Safe and improved drugs, accelerated drug discovery, better diagnostic and quick methods for detecting an infection either active or latent, development of new and safe vaccines, and completely novel classes of therapeutics and other medical applications are added feathers in its cap. The technology has revolutionized understanding of diseases as diverse as cystic fibrosis and cancer. Pharmaceutical biotechnology being one of the important sectors involves using animals or hybrids of tumor cells or leukocytes or cells ( prokaryotic, mammalian) to produce safer, more efficacious, and cost-effective versions of conventionally produced biopharmaceuticals. The launch of the new biopharmaceutical or drug requires screening and development. Mice were widely used as research animals for screening. However, in the wake of animal protection, animal cell culture offers accurate and inexpensive source of cells for drug screening and efficacy testing. Pharmaceutical biotechnology’s greatest potential lies in gene therapy and stem cell-based therapy. The underlying cause of defect of many inherited diseases is now located and characterized. Gene therapy is the insertion of the functional gene in place of defective gene into cells to prevent, control, or cure disease. It also involves addition of genes for pro-drug or cytokines to eliminate or suppress the growth of the tumors in cancer treatment.

But the progress so far is viewed by many scientists as only a beginning. They believe that, in the not-so-distant future, the refinement of “targeted therapies” should dramatically improve drug safety and efficacy. The development of predictive technologies may lead to a new era in disease prevention, particularly in some of the world’s rapidly developing economies. Yet the risks cannot be ignored as new developments and discoveries pose new questions, particularly in areas as gene therapy, the ethics of stem cell research, and the misuse of genomic information.

Many bio-therapeutic agents in clinical use are biotech pharmaceuticals. Insulin was among the earliest recombinant drugs. Canadian physiologists Frederick Banting and Charles Best discovered and isolated insulin in 1921. In that time many patients diagnosed with diabetes died within a few years. In the mid-1960s, several groups reported synthesizing the hormone.

The first “bioengineered” drug, a recombinant form of human insulin, was approved by the US Food and Drug Administration (FDA) in 1982. Until then, insulin was obtained from a limited supply of beef or pork pancreas tissue. By inserting the human gene for insulininto bacteria, scientists were able to achieve lifesaving insulinproduction in large quantities. In the near future, patients with diabetes may be able to inhale insulin, eliminating the need for injections. Inhaled insulinproducts like Exubera® were approved by the USFDA; however, it was pulled out and other products like Technosphere® insulin (Afrezza®) are under investigation. They may provide relief from prandial insulin. Afrezza consists of a pre-meal insulinpowder loaded into a cartridge for oral inhalation.

Technosphere technology: The technology allows administration of therapeutics through pulmonary route which otherwise were required to be given as injections. These formulations have broad spectrum of physicochemical characteristics and are prepared with a diverse assortment of drugs with protein or small molecule which may be hydrobhobic or hydrophilic or anionic or cationic in nature. The technology can have its applicability not only through pulmonary route but also for other routes of administration including local lung delivery.

The first recombinant vaccine, approved in 1986, was produced by cloning a gene fragment from the hepatitis B virus into yeast (Merck’s Recombivax HB). The fragment was translated by the yeast’s genetic machinery into an antigenic protein. This was present on the surface of the virus that stimulates the immune response. This avoided the need to extract the antigen from the serum of people infected with hepatitis B.

The Food and Drug administration (FDA) approved more biotech drugs in 1997 than in the previous several years combined. The FDA has approved many recombinant drugs for human health conditions. These include AIDS, anemia, cancers (Kaposi’s sarcoma, leukemia, and colorectal, kidney, and ovarian cancers), certain circulatory problems, certain hereditary disorders (cystic fibrosis, familial hypercholesterolemia, Gaucher’s disease, hemophilia A, severe combined immunodeficiency disease, and Turner’s syndrome), diabetic foot ulcers, diphtheria, genital warts, hepatitis B, hepatitis C, human growth hormone deficiency, and multiple sclerosis. Today there are more than 100 recombinant drugs and vaccines. Because of their efficiency, safety, and relatively low cost, molecular diagnostic tests and recombinant vaccines may have particular relevance for combating long-standing diseases of developing countries, including leishmaniasis (a tropical infection causing fever and lesions) and malaria.

Stem cell research is very promising and holds tremendous potential to treat neurodegenerative disorders, spinal cord injuries, and other conditions related to organ or tissue loss.

DNA analysis is another powerful technique which compares DNA pattern [ 14 ] after performing RFLP and probing it by minisatellite repeat sequence between two or more individuals. Its modification, DNA profiling (process of matching the DNA profiles for STS markers in two or more individuals; see chapter 18), which utilizes multilocus PCR analysis of DNA of suspect and victims, is very powerful and is useful in criminal investigation, paternity disputes, and so many other legal issues. The analysis is very useful in criminal investigations and involves evaluation of DNA from samples of the hair, body fluids, or skin at a crime scene and comparison of these with those obtained from the suspects.

Improved Diagnostic and Therapeutic Capabilities

The sequencing of the human genome in 2003, has given scientists an incredibly rich “parts list” with which to better understand why and how disease happens. It has given added power to gene expression profiling, a method of monitoring expression of thousands of genes simultaneously on a glass slide called a microarray. This technique can predict the aggressiveness of cancer.

The development of monoclonal antibodies in 1975 led to a medical revolution. The body normally produces a wide range of antibodies—the immune system proteins—that defend our body and eliminate microorganisms and other foreign invaders. By fusing antibody-producing cells with myeloma cells, scientists were able to generate antibodies that would, like “magic bullets,” bind with specific targets including unique markers, called antigenic determinants ( epitopes), on the surfaces of inflammatory cells. When tagged with radioisotopes or other contrast agents, monoclonal antibodies can help in detecting the location of cancer cells, thereby improving the precision of surgery and radiation therapy and showing—within 48 h—whether a tumor is responding to chemotherapy.

The polymerase chain reaction, a method for amplifying tiny bits of DNA first described in the mid-1980s, has been crucial to the development of blood tests that can quickly determine exposure to the human immunodeficiency virus (HIV). Genetic testing currently is available for many rare monogenic disorders, such as hemophilia, Duchenne muscular dystrophy, sickle cell anemia, thalassemia, etc.

Another rapidly developing field is proteomics, which deals with analysis and identification of proteins. The analysis is done by two-dimensional gel electrophoresis of the sample and then performing mass spectrometric analysis for each individual protein. The technique may be helpful in detecting the disease-associated protein in the biological sample. They may indicate early signs of disease, even before symptoms appear. One such marker is C-reactive protein, an indicator of inflammatory changes in blood vessel walls that presage atherosclerosis.

Nanomedicine is a rapidly moving field. Scientists are developing a wide variety of nanoparticles and nanodevices, scarcely a millionth of an inch in diameter, to improve detection of cancer, boost immune responses, repair damaged tissue, and thwart atherosclerosis. The FDA has approved a paclitaxel albumin-stabilized nanoparticle formulation (Abraxane® for injectable suspension, made by Abraxis BioScience) for the treatment of metastatic adenocarcinoma of the pancreas. Nanoparticles are being explored in heart patients in the USA as a way to keep their heart arteries open following angioplasty.

Therapeutic proteins are those, which can replace the patients naturally occurring proteins, when levels of the natural proteins are low or absent due to the disease. High-throughput screening, conducted with sophisticated robotic and computer technologies, enables scientists to test tens of thousands of small molecules in a single day for their ability to bind to or modulate the activity of a “target,” such as a receptor for a neurotransmitter in the brain. The goal is to improve the speed and accuracy of therapeutic protein or potential drug discovery while lowering the cost and improving the safety of pharmaceuticals that make it to market.

Many of the molecules utilized for detection also have therapeutic potential too, for example, monoclonal antibodies. The monoclonal antibodies are approved for the treatment of many diseases as cancer, multiple sclerosis, and rheumatoid arthritis. They are currently being tested in patients as potential treatments for asthma, Crohn’s disease, and muscular dystrophy. As the antibodies may be efficiently targeted against a particular cell surface marker, thus they are used to deliver a lethal dose of toxic drug to cancer cells, avoiding collateral damage to nearby normal tissues.

Agricultural Biotechnology

The manhas made tremendous progress in crop improvement in terms of yield; still the ultimate production of crop is less than their full genetic potential. There are many reasons like environmental stresses (weather condition as rain, cold, frost), diseases, pests, and many other factors which reduce the ultimate desired yield affecting crop productivity. The efforts are going on to design crops which may be grown irrespective of their season or can be grown in frost or drought conditions for maximum utilization of land, which would ultimately affect crop productivity [ 24 ]. Agricultural biotechnology aims to introduce sustainable agriculturalpractices with best yield potential and minimal adverse effects on environment (Fig. 1.7 ). For example, combating pests was a major challenge. Thus, the gene from bacterium , the Bt gene, that functions as insect-resistant gene when inserted into crop plants like cotton, corn, and soybean helps prevent the invasion of pathogen, and the tool is called . This management is helpful in reducing usage of potentially dangerous pesticides on the crop. Not only the minimal or low usage of pesticides is beneficial for the crop but also the load of the polluting pesticides on environment is greatly reduced [ 24 ].

Various applications of agricultural biotechnology

Resistance to Infectious Agents Through Genetic Engineering

• The gene comes from the soil bacterium .
• The gene produces crystal proteins called Cry proteins. More than 100 different variants of the Bt toxins have been identified which have different specificity to target insect lepidoptera. For eg., CryIa for butterflies and CRYIII for beetles.
• These Cry proteins are toxic to larvae of insects like tobacco budworm, armyworm, and beetles.
• The Cry proteins exist as an inactive protoxins.
• These are converted into active toxin in alkaline pH of the gut upon solubilization when ingested by the insect.
• After the toxin is activated, it binds to the surface of epithelial cells of midgut and creates pores causing swelling and lysis of cells leading to the death of the insect (larva).
• The genes (cry genes) encoding this protein are isolated from the bacterium and incorporated into several crop plants like cotton, tomato, corn, rice, and soybean.

The proteins encoded by the following cry genes control the pest given against them:

• Cry I Ac and cry II Ab control cotton bollworms.
• Cry I Ab controls corn borer.
• Cry III Ab controls Colorado potato beetle.
• Cry III Bb controls corn rootworm.
• A nematode infects tobacco plants and reduces their yield.
• The specific genes (in the form of cDNA) from the parasite are introduced into the plant using -mediated transformation.
• The genes are introduced in such a way that both sense/coding RNA and antisense RNA (complimentary to the sense/coding RNA) are produced.
• Since these two RNAs are complementary, they form a double-stranded RNA (ds RNA).
• This neutralizes the specific RNA of the nematode, by a process called RNA – interference.
• As a result, the parasite cannot multiply in the transgenic host, and the transgenic plantis protected from the pest.

These resistant crops result in reduced application of pesticides. The yield is high without the pathogen infestations and insecticides. This also helps to reduce load of these toxic chemicals in the environment.

The transformation techniques and their applications are being utilized to develop rice, cassava, and tomato, free of viral diseases by “International Laboratory for Tropical Agricultural Biotechnology” (ILTAB). ILTAB in 1995 reported the first transfer of a resistance gene from a wild-type species of rice to a susceptible cultivated rice variety. The transferred gene expressed and imparted resistance to crop-destroying bacterium Xanthomonas oryzae . The resistant gene was transferred into susceptible rice varieties that are cultivated on more than 24 million hectares around the world [ 6 ].

The recombinant DNA technology reduces the time between the identification of a particular gene to its application for betterment of crops by skipping the labor-intensive and time-consuming conventional breeding [ 3 ]. For example, the alteration of known gene in plant for the improvement of yield or tolerance to adverse environmental conditions or resistance to insect in one generation and its inheritance to further generations. Plant cell and tissue culture techniques are one of the applications where virus-free plants can be grown and multiplied irrespective of their season on large scale (micropropogation), raising haploids, or embryo rescue. It also opens an opportunity to cross two manipulated varieties or two incompatible varieties (protoplast culture) for obtaining best variety for cultivation.

With the help of technology, new, improved, and safe agricultural products may emerge which would be helpful for maintaining contamination-free environment. Biotechnology has the potential to produce:

• High crop yields [ 4 ]
• Crops are engineered to have desirable nutrients and better taste (e.g., tomatoes and other edible crops with increased levels of vitamin C, vitamin E, and/or beta-carotene protect against the risk of some prevalent chronic diseases and rice with increased iron levels protects against anemia)
• Insect- and disease-resistant plants
• Genetic modification avoids nonselective changes
• Longer shelf life of fruits and vegetables

The potential of biotechnology may contribute to increasing agricultural, food, and feed production, protecting the environment, mitigating pollution, sustaining agricultural practices, and improving human and animal health. Some agricultural crops as corn and marine organisms can be potential alternative for biofuel production. The by-products of the process may be processed to produce other chemical feedstocks for various products. It is estimated that the world’s chemical and fuel demand could be supplied by such renewable resources in the first half of the next century [ 5 ].

Food Biotechnology

Food biotechnology is an emerging field, which can increase the production of food, improving its nutritional content and improving the taste of the food. The food is safe and beneficial as it needs fewer pesticides and insecticides. The technology aims to produce foods which have more flavors, contain more vitamins and minerals, and absorb less fat when cooked. Food biotechnology may remove allergens and toxic components from foods, for their better utility [ 6 , 7 ].

Environmental Biotechnology

Environmental biotechnology grossly deals with maintenanceof environment, which is pollution-free, the water is contamination-free, and the atmosphere is free of toxic gases. Thus, it deals with waste treatment, monitoring of environmental changes, and pollution prevention. Bioremediation in which utilization of higher living organisms (plants: phytoremediation) or certain microbial species for decontamination or conversion of harmful products is done is the main application of environmental biotechnology. The enzyme bioreactors are also being developed which would pretreat some industrial and food waste components and allow their removal through the sewage system rather than through solid waste disposal mechanisms. The production of biofuel from waste can solve the fuel crisis (biogas). Microbes may be engineered to produce enzymes required for conversion of plant and vegetable materials into building blocks for biodegradable plastics. In some cases, the by-products of the pollution-fighting microorganisms are themselves useful. For example, methane can be derived from a form of bacteria that degrades sulfur liquor, a waste product of paper manufacturing. This methane thus obtained is used as a fuel or in other industrial processes. Insect- and pest-resistant crops have reduced the use and environmental load of insecticides and pesticides. Insect-protected crops allow for less potential exposure of farmers and groundwater to chemical residues while providing farmers with season-long control.

Industrial Biotechnology

The utilizationof biotechnological tools (bioprocessing) for the manufacturing of biotechnology-derived products (fuels, plastics, enzymes, chemicals, and many more compounds) on industrial scale is industrial biotechnology. The aim is to develop newer industrial manufacturing processes and products, which are economical and better than preexisting ones with minimal environmental impact. In industrial biotechnology, (1) microorganisms are being explored for producing material goods like fermentation products as cheese; (2) biorefineries where oils, sugars, and biomass may be converted into biofuels, bioplastics, and biopolymers; (3) and value-added chemicals from biomass. The utilization of modern techniques can improve the efficiency and reduces the environmental impacts of industrial processes like textile, paper, pulp, and chemical manufacturing. For example, development and usage of biocatalysts, such as enzymes, to synthesize chemicals and development of antibiotics and better tasting liquors and their usage in food industry have provided safe and effective processing for sustainable productions. Biotechnological tools in the textile industry are utilized for the finishing of fabrics and garments. Biotechnology also produces spider silk and biotech-derived cotton that is warmer and stronger and has improved dye uptake and retention, enhanced absorbency, and wrinkle and shrink resistance.

Biofuels may be derived from photosynthetic organisms, which capture solar energy, transform it in other products like carbohydrates and oils, and store them. Different plants can be used for fuel production:

• Bioethanol can be obtained from sugar (as sugarcane or sugar beet) or starch (like corn or maize). These are fermented to produce ethanol, a liquid fuel commonly used for transportation.
• Biodiesel can be obtained from natural oils from plants like oil palm, soybean, or algae. They can be burned directly in a diesel engine or a furnace, or blended with petroleum, to produce fuels such as biodiesel.
• Wood and its by-products can be converted into liquid biofuels, such as methanol or ethanol, or into wood gas. Wood can also be burned as solid fuel, like the irewood.

In these kinds of biological reaction, there are many renewable chemicals of economic importance coproduced as side streams of bioenergy and biofuels as levulinic acid, itaconic acid, and sorbitol. These have tremendous economic potential and their fruitful usage would depend upon the collaboration for research and development between the government and the private sector.

Enzyme Production

The enzymeshave big commercial and industrial significance. They have wide applications in food industry, leather industry, pharmaceuticals, chemicals, detergents, and research. In detergents the alkaline protease, subtilisin (from Bacillus subtilis ), was used by Novo Industries, Denmark. The production of enzymes is an important industrial application with world market of approximately 5 billion dollars. The enzymes can be obtained from animals, plants, or microorganisms. The production from microorganisms is preferred as they are easy to maintain in culture with simple media requirements and easy scale-up. The important enzymes for the industrial applications are in food industry, human application, and research. A few animal enzymes are also important as a group of proteolytic enzymes, for example, plasminogen activators, which act on inactive plasminogen and activate it to plasmin, which destroys fibrin network of blood clot. Some of the plasminogen activators are urokinase and tissue plasminogen activators (t-PA). Urokinase (from urine) is difficult to obtain in ample quantity; thus, t-PA is obtained from cells grown in culture medium. Streptokinase (bacterial enzyme) is also a plasminogen activator but is nonspecific and immunogenic.

Enzyme engineering is also being tried where modifications of specific amino acid residue are done for improving the enzyme properties. One of the enzymes chymosin (rennin) coagulates milk for cheese manufacturing.

The enzymes can be produced by culturing cells, growing them with appropriate substrates in culture conditions. After optimum time the enzymes may be obtained by cell disruption (enzymatic/freeze–thaw/osmotic shock) followed by preparative steps (centrifugation, filtration), purification, and analysis. The product is then packaged and ultimately launched in the market.

After their production, they can be immobilized on large range of materials (agar, cellulose, porous glass, or porous alumina) for subsequent reuse. Some of the important industrial enzymes are α-amylase (used for starch hydrolysis), amyloglucosidase (dextrin hydrolysis), β-galactosidase (lactose hydrolysis), aminoacylase (hydrolysis of acylated L-amino acids), glucose oxidase (oxidation of glucose), and luciferase (bioluminescence). Some of the medically important enzymes are urokinase and t-PA for blood clot removal and L-asparaginase for removal of L-asparagine essential for tumor growth and thus used for cancer chemotherapy in leukemia.

Exploring Algae for Production of Biofuels

The energyrequirement of present population is increasing and gradually fossil fuels are rapidly depleting. Thus, renewable energy sources like solar energy and wind-, hydro-, and biomass-based energy are being explored worldwide. One of the feedstocks may be microalgae, which are fast-growing, photosynthetic organisms requiring carbon dioxide, some nutrients, and water for its growth. They produce large amount of lipids and carbohydrates, which can be processed into different biofuels and commercially important coproducts. The production of biofuels using algal biomass is advantageous as they (1) can grow throughout the year and thus their productivity is higher than other oil seed crops, (2) have high tolerance to high carbon dioxide content, (3) utilize less water, (4) do not require herbicides or pesticides with high growth potential (waste water can be utilized for algal cultivation), (5) can sustain harsh atmospheric conditions, and (6) do not interfere with productivity of conventional crops as they do not require agricultural land. The production of various biofuels from algae is schematically represented in Fig. 1.8 .

Different biofuel productions by using microalgae. The algae use sunlight, CO2, water, and some nutrients

Algae can serve as potential source for biofuel production; however, biomass production is low. The production has certain limitations, as cultivation cost is high with requirement of high energy [ 1 ].

Marine or Aquatic Biotechnology

Marine or aquatic biotechnology also referred to as “blue biotechnology” deals with exploring and utilizing the marine resources of the world. Aquatic or marine life has been intriguing and a source of livelihood for many since years. As major part of earth is acquired by water, thus nearly 75–80 % types of life forms exist in oceans and aquatic systems. It studies the wide diversity found in the structure and physiology of marine organisms. They are unique in their own ways and lack their equivalent on land. These organisms have been explored and utilized for numerous applications as searching new treatment for cancer or exploring other marine resources, because of which the field is gradually gaining momentum and economic opportunities [ 19 ]. The global economic benefits are estimated to be very high. The field aims to:

• Fulfill the increasing food supply needs
• Identify and isolate important compounds which may benefit health of humans
• Manipulate the existing traits in sea animals for their improvement
• Protect marine ecosystem and gain knowledge about the geochemical processes occurring in oceans

Some of the major applications are discussed:

• Aquaculture: Aquaculture refers to the growth of aquatic organisms in culture condition for commercial purposes. These animals may be shellfish, finfish, and many others. Mariculture refers to the cultivation of marine animals. Their main applications are in food, food ingredients, pharmaceuticals, and fuels, the products are in high demand, and various industries are in aquaculture business, for example, crawfish farming (Louisiana), catfish industry (Alabama and Mississippi Delta), and trout farming (Idaho and West Virginia).
• Transgenic species of salmon with growth hormone gene has accelerated growth of salmons.
• Molt-inhibiting (MIH) from blue crabs leads to soft-shelled crab.
• : Anovel protein antifreeze protein (AFP) was identified. AFPs were isolated from Northern cod (bottom-dwelling fish) living at the Eastern Canada coast and teleosts living in extremely cold weather of Antarctica. AFPs have been isolated from Osmerus mordax (smelt), Clupea harengus (herring), Pleuronectes americanus (winter flounder), and many others. Due to antifreeze properties (lowering the minimal freezing temperature by 2–3 °C), the gene has potential for raising plants which are cold tolerant (e.g., tomatoes).
• Medicinal applications : For osteoporosis, salmon calcitonin (calcitonin is thyroid hormone promoting calcium uptake and bone calcification) with 20 times higher bioactivity is available as injection and nasal spray.
• Hydroxyapatite ( HA ): Obtained from coral reefs and is an important component of bone and cartilage matrix. Its implants are prepared by Interpore Internationals which may be used for filling gaps in fractured bones.

Many anti-inflammatory, analgesic, anticancerous compounds have been identified from sea organisms which can have tremendous potential for human health.

Tetrodotoxin (TTX) is the most toxic poison (10,000 times more lethal than cyanide) produced by Japanese pufferfish or blowfish ( Fugu rubripes ). TTX is being used to study and understand its effect on sodium channels which can help guide the development of drugs with anesthetic and analgesic properties.

Other Products

• Taq polymerase from Thermus aquaticus which is used in PCR reactions and obtained from hot spring Archaea.
• Collagenase (protease) obtained from Vibrio is used in tissue engineering and culturing.

Transgenic Animals and Plants

In the early1980s, inserting DNA from humans into mice and other animals became possible. The animals and plants which have foreign gene in each of their cells are referred to as transgenic organisms and the inserted gene as transgene. Expression of human genes in these transgenic animals can be useful in studies, as models for the development of diabetes, atherosclerosis, and Alzheimer’s disease. They also can generate large quantities of potentially therapeutic human proteins. Transgenic plants also offer many economic, safe, and practical solutions for production of variety of biopharmaceuticals. The plants have been engineered to produce many blood products (human serum albumin, cytokines), human growth hormone, recombinant antibodies, and subunitvaccines.

The usage of transgenic plants for the production of recombinant pharmaceuticals might open new avenues in biotechnology. As plants can be grown inexpensively with minimal complicated requirements, thus they may have tremendous therapeutic potential. The plants have been engineered to produce more nutrients or better shelf life. The transgenic plants have been created which have genes for insect resistance (Bt cotton, soybean, corn). Now billion acres of land is used for cultivation of genetically engineered crops of cotton, corn, and soybean as they have higher yield and are pest resistant. However, due to social, ethical, and biosafety issues, they have received acceptance as well as rejections at many places and health and environment-related concerns in many parts of the world [ 8 ].

Response to Antibiotic Resistance

Antibiotics areone of the broadly used therapeutic molecules produced by certain classes of microorganisms (bacteria and fungi) which can be used in diverse clinical situations to eliminate bacteria, improve symptoms, and prevent number of infections. Antibiotics have various other applications apart from clinical aspects. They can be used for the treatment of tumors and treatment of meat, in cattles and livestocks, in basic biotechnological work. However, their effectiveness is a matter of concern as bacteria which are continuously exposed to certain antibiotics might become resistant to it due to accumulation of mutations. These days antibiotic-resistant bacteria have increased and some of them have developed multiple drug resistance. Thus, it has become very difficult to initiate therapy in diseases like tuberculosis and leprosy. Biotechnology is solving the urgent and growing problem of antibiotic resistance. With the help of bioinformatics—powerful computer programs capable of analyzing billions of bits of genomicsequence data—scientists are cracking the genetic codes of bacteria and discovering “weak spots” vulnerable to attack by compounds identified via high-throughput screening. This kind of work led in 2000 to the approval of Zyvox (linezolid), an antibiotic to reach the market in 35 years.

Lytic bacteriophage viruses that infect and kill bacteria may be another way to counter resistance. First used to treat infection in the 1920s, “phage therapy” was largely eclipsed by the development of antibiotics. However, researchers in the former Soviet Republic of Georgia reported that a biodegradable polymer impregnated with bacteriophages and the antibiotic Cipro successfully healed wounds infected with a drug-resistant bacterium.

After exposure of strontium-90, three Georgian lumberjacks from village Lia had systemic effects, and two of them developed severe local radiation injuries which subsequently became infected with Staphylococcus aureus . Upon hospitalization, the patients were treated with various medications, including antibiotics and topical ointments; however, wound healing was only moderately successful, and their S. aureus infection could not be eliminated. Approximately 1 month after hospitalization, treatment with PhagoBioDerm (a wound-healing preparation consisting of a biodegradable polymer impregnated with ciprofloxacin and bacteriophages) was initiated. Purulent drainage stopped within 2–7 days. Clinical improvement was associated with rapid (7 days) elimination of the etiologic agent, and a strain of S. aureus responsible for infection was resistant to many antibiotics (including ciprofloxacin) but was susceptible to the bacteriophages contained in the PhagoBioDerm preparation [ 11 ].

The Challenges for the Technology

Gene therapy.

Some biotechapproaches to better health have proven to be more challenging than others. An example is gene transfer, where the defective gene is replaced with a normally functioning one. The normal gene is delivered to target tissues in most cases by virus that is genetically altered to render it harmless. The first ex vivo gene transfer experiment, conducted in 1990 at the National Institutes of Health (NIH), on Ashanti DeSilva who was suffering from severe combined immunodeficiency (SCID) helped boost her immune response and successfully corrected an enzyme deficiency. However, treatment was required every few months. However, 9 years later, a major setback occurred in gene therapy trial after the death of 18-year-old Jesse Gelsinger suffering from ornithine transcarbamylase (OTC) deficiency due to intense inflammatory responses followed by gene therapy treatment. There were some positive experiences and some setbacks from gene therapy trials leading to stricter safety requirements in clinical trials.

Designer Babies

The fancyterm designer baby was invented by media. Many people in society prefer embryos with better traits, intellect, and intelligence. They want to select embryo post germline engineering. This technique is still in infancy but is capable of creating lot of differences in the society thus requires appropriate guidelines.

Genetically Modified Food

Genetically modifiedcrops harboring genes for insect resistance were grown on billion of acres of land. These crops became very popular due to high yield and pest resistance. However, some of the pests gradually developed resistance for a few of these transgenic crops posing resistant pest threat. The other technologies as “traitor” and “terminator” technologies pose serious risk on crop biodiversity and would impart negative characters in the crop (they were not released due to public outcry).

Pharmacogenomics

Scientists do not believe they will find a single gene for every disease. As a result, they are studyingrelationships between genes and probing populations for variations in the genetic code, called single nucleotide polymorphisms, or SNPs, that may increase one’s risk for a particular disease or determine one’s response to a given medication. This powerful ability to assign risk and response to genetic variations is fueling the movement toward “individualized medicine.” The goal is prevention, earlier diagnosis, and more effective therapy by prescribing interventions that match patients’ particular genetic characteristics.

Tissue Engineering

Tissue engineering is one of the emerging fields with tremendous potential to supply replacement tissue and organ option for many diseases. Lot is achieved, lot more need to be achieved.

Ethical Issues

The pursuit of cutting-edge research “brings us closer to our ultimate goal of eliminating disability and disease through the best care which modern medicine can provide.” Understanding of the genetics of heart disease and cancer will aid the development of screening tools and interventions that can help prevent the spread of these devastating disorders into the world’s most rapidly developing economies.

Biotechnology is a neutral tool; nevertheless, its capabilities raise troubling ethical questions. Should prospective parents be allowed to “engineer” the physical characteristics of their embryos? Should science tinker with the human germ line, or would that alter in profound and irrevocable ways what it means to be human?

More immediately, shouldn’t researchers apply biotechnology—if they can—to eliminate health disparities among racial and ethnic groups? While genetic variation is one of many factors contributing to differences in health outcome (others include environment, socioeconomic status, health-care access, stress, and behavior), the growing ability to mine DNA databases from diverse populations should enable scientists to parse the roles these and other factors play.

Biotechnology along with supportive health-care infrastructure can solve complicated health problems. Accessibility to the new screening tests, vaccines, and medications and cultural, economic, and political barriers to change must be overcome. Research must include more people from disadvantaged groups, which will require overcoming long-held concerns, some of them have had about medical science.

Biotechnology has been a significant force which has improved the quality of lives and has incalculably benefitted human beings. However, technology does have prospects of doing harm also due to unanticipated consequences. Each technology is subjected to ethical assessment and requires a different ethical approach. Obviously the changes are necessary as technology can have major impact on the world; thus, a righteous approach should be followed. There is uncertainty in predicting consequences, as this powerful technology has potential to manipulate humans themselves. Ethical concerns are even more important as the future of humanity can change which require careful attention and consideration. Therefore, wisdom is required to articulate our responsibilities toward environment, animals, nature, and ourselves for the coming future generations. We need to differentiate what is important technologically rather that what technology can do. For an imperative question, that is, whether this can be achieved, the research must answer “Why should it be achieved”? Who would it benefit?

Issues Related to Safety

• As the new GM crops are entering the market, the issue regarding their consumption, whether they are safe, without any risk, is one of the important concerns [ 2 ]. Though the results related to safety and usage are well reported (as compared to conventional crops), unknown fear from these products makes them non acceptable at many places [ 20 ].
• As insect- and pest-resistant varieties are being prepared and used as Bt genes in corn and cotton crops, there exists a risk of development of resistance insect population. Another important factor is that these resistant crops may harm other species like birds and butterfly.
• The development of more weeds may occur as cross-pollination might result in production of weeds with herbicide resistance which would be difficult to control.
• The gene transfers might cross the natural species boundary and affect biological diversity.
• The judgment of their usage would depend upon the clear understanding of risks associated with safety of these products in determining the impact of these on environment, other crops, and other animal species.

Future of the Technology

With the understanding of science, we should understand that genetic transfers have been occurring in animals and plant systems; thus, the risk of the biotechnology-derived products is similar as conventional crops [ 12 ].

The biotechnology products would be acceptable to many if they are beneficial and safe. People are willing to buy crops free of pesticides and insecticides. Nowadays people are also accepting crops grown without the usage of chemical fertilizers or pesticides, which are high in nutritive values.

The labeling of the product is also an ethical issue as some believe that labeling any product as biotechnology product might be taken by consumer as warning signs; however, others believe that labeling should be done as consumer has every right to know what he is consuming [ 9 ]. The products may be acceptable if consumers can accept the food derived from biotechnology weighing all pros and cons and, if the price is right, has more nutritive values, is good in taste, and is safe to consume [ 10 ].

Biotechnology is at the crossroads in terms of fears and thus public acceptance [ 15 ]. Surprisingly the therapeutic products are all accepted and find major place in biopharmaceutical industry, but food crops are still facing problems in worldwide acceptance. The future of the world food supply depends upon how well scientists, government, and the food industry are able to communicate with consumers about the benefits and safety of the technology [ 13 , 16 ]. Several major initiatives are under way to strengthen the regulatory process and to communicate more effectively with consumers by conducting educational programs [ 18 , 23 ].

Chapter End Summary

• The advantages of biotechnology are so broad that it is finding its place in virtually every industry. It has applications in areas as diverse as pharmaceuticals, diagnostics, textiles, aquaculture, forestry, chemicals, household products, environmental cleanup, food processing, and forensics to name a few.
• Biotechnology is enabling these industries to make new or better products, often with greater speed, efficiency, and flexibility.
• With the applications of recombinant DNA technology, more safer and therapeutic drugs are produced. These recombinant products do not elicit unwanted immunological response which is observed when the product is obtained from other live or dead sources. Many of these therapeutics are approved for human usage, and many of them are in the phase of development.
• Immunological and DNA-based techniques like PCR (polymerase chain reaction) are used for early diagnosis of disorders. PCR and NAAT with microarray can be utilized for the diagnosis of many diseases, and it can detect mutations in gene.
• The technology holds promise through stem cell research and gene therapy and holds applications in forensic medicine.
• The technique may be helpful in developing useful and beneficial plants. It overcomes the limitations of traditional plant breeding. The techniques of plant tissue culture, transgenics, and marker-assisted selections are largely used for selecting better yielding varieties and imparting quality traits in plants.
• Food industries. Production of single-cell protein, spirulina, enzymes, and solid-state fermentations
• Increase and improvement of agricultural production
• Production of therapeutic pharmaceuticals
• Production of vaccines and monoclonal antibodies
• Cultivation of virus for vaccine production

Multiple Choice Questions

• All of the above
• Vitamin D and calcium
• Growth hormone
• Tissue plasminogen activator
• Factor VIII
• Genetically modifying organism
• Production of therapeutics
• Production of better diagnosis
• Increase in yield of crops
• Improved crop varieties
• Lesser fertilizers and agrochemicals
• All of these
• It is resistant to it.
• The toxin is enclosed in vesicle.
• The toxin is present in inactive form.
• None of these.
• Gene therapy
• Replacement protein therapy
• Stem cell therapy
• The productivity would improve.
• The usage of chemical agent would be reduced.
• The environment and crop would be insecticide free.
• All of the above.
• Detoxifying waste material
• Burying waste material
• Burning waste material
• None of these

(1) In all the cells of our body, all the genes are active.

(2) In different cells of our body, different genes are active.

(3) Gene expression is spatially and temporally regulated.

• All 1, 2, and 3 are correct.
• 1 and 2 are correct.
• 1 and 3 are correct.
• 2 and 3 are correct.
• Inoculation with monoclonal antibody was able to prevent small pox.
• Inoculation with pus from sores due to cowpox could prevent small pox.
• Attenuated vaccine was able to prevent small pox.
• None of the above.
• 1. (c); 2. (a); 3. (c); 4. (d); 5. (d); 6. (d); 7. (c); 8. (a); 9. (d); 10. (a); 11. (d); 12. (b)

Review Questions

• Q1. What are cry proteins? What is their importance?
• Q2. Give some applications of biotechnology in agriculture.
• Q3. What is your opinion about labeling of biotechnology-based food product as rDNA technology derived product?
• Q4. What are applications of biotechnology in maintaining environment?
• Q5. What is medical biotechnology?
• Q6. What are the challenges faced by biotechnology industry?
• Q7. What do you think about GM crops?

Some Related Resources

• http://ificinfo.health.org/backgrnd/BKGR14.htm
• http://www.bio.org/aboutbio/guide1.html
• http://www.bio.org/aboutbio/guide2000/guide00_toc.html
• http://www.bio.org/aboutbio/guide3.html
• http://www.bio.org/aboutbio/guide4.html
• http://www.dec.ny.gov/energy/44157.html
• http://www.ers.usda.gov/whatsnew/issues/biotech/define.htm
• http://www.nal.usda.gov/bic/bio21
• http://www.nature.com/nbt/press_release/nbt1199.html
• www.angelfire.com/scary/intern/links.html
• www.bio-link.org/library.htm
• www.biospace.com
• www.dnai.org
• www.fiercebiotech.com
• www.iastate.edu
• www.icgeb.trieste.it
• www.ncbi.nlm.nih.gov

Hot Research Topics in Biotech in 2022

The past few years years have seen leaps and strides of innovation as scientists have worked to develop and produce new mRNA vaccinations and made major developments in biotech research. During this time, they’ve also faced challenges. Ongoing supply chain disruptions , the Great Resignation, and the pandemic have impacted biotech labs and researchers greatly, forcing lab managers and PIs to get creative with lab supply purchasing, experiment planning, and the use of technology in order to maintain their research schedules.

“The pace of innovation specific to COVID to be able to develop both medicines related to antibodies as well as vaccines is just staggering. Those of us in the industry are in awe of the innovation we’re witnessing on a daily basis. We’ve been behind in the use of automation, software, and AI that can make our industry more efficient — that’s where we’re headed,” says Michelle Dipp, Cofounder and Managing Partner, Biospring Partners on the This is ZAGENO podcast .

At the start of 2022, current biotech research projects are exploring advancements in medicine, vaccines, the human body and treatment of disease, bacteria and immunology, and viruses like the Coronavirus that affected the globe in ways we couldn’t have anticipated.

Biotech Research Processes are Changing

As Michelle explained, the research that’s happening is changing, and so is the way that scientists conduct it. Influenced by both B2C ecommerce and the growing dependence on remote and cloud-based working, biotech labs are undergoing digital transformations . This means more software, AI, and automation in the lab, along with modern digital procurement strategies and integrated systems for lab operations.

Here are some of the top biotech research trends and recent biotech research papers that are changing the world of science and leading to innovation in life sciences.

Top 6 Biotech Research Topics for 2022

Science journals have never been more popular as they’ve been in the past several years. Resonating with the general public, biotech research papers have found their way into the hands and social media streams of interested citizens and scientists alike.

As we look to the most credible, peer-reviewed sources for recent innovations like PubMed , the Journal of Biotechnology , BioTech , and Biotechnology Journal , the trending themes in biotech research are in direct response to COVID-19, like vaccine development, respiratory virus research, and RNA-based pharmaceuticals. Additionally, there have been major advances in metabolism and the human microbiome, as well as further exploration in microvesicles.

All of the research happening has the potential to change millions of people’s quality of life, prevent and treat illnesses that currently have high mortality rates, and change healthcare around the world.

Here's what's happening in biotech research.

1. Vaccine Development mRNA vaccine development has been in the works since 1989 and was accelerated in recent years to combat the global COVID-19 pandemic. Researchers like Maruggi, Zhang, Li, Ulmer, Yu and their team believe that mRNA vaccines could change infectious disease control as we know it as a prophylactic means of disease prevention for diseases like HIV, Zika, and the flu.

Recent developments in mRNA research from Pardi, Hogan, and Weissman in 2020 explored the ways that mRNA vaccines can combat certain cancers and infectious pathogens that were previously resistant to existing vaccine options.

With new access to data from the 3.4 billion+ COVID-19 mRNA vaccines that have been administered worldwide, researchers have been able to determine the risks associated with mRNA vaccines , which brings forward new topics for research in the medical and pharmaceutical sides of the biotech industry. mRNA vaccines are faster to develop and can help prevent more diseases than traditional vaccine methods.

2. Respiratory Viruses Acute respiratory diseases (ARDs) like those caused by the SARS-CoV pathogen or the influenza virus lead to morbidity and mortality, and can lead to pneumonia, which can be fatal for immunocompromised or elderly patients — they represent a huge impact to human society.

Identifying the cause of ARDs and identifying viral infections from COVID-19 has become an issue of public health and safety, leading research groups like Zhang, Wang, and team to seek out more accurate and faster ways to detect respiratory viruses .

Understanding these respiratory virus mechanisms can help lead to better protection, prevention, and treatments for respiratory viruses, which have a mortality rate of up to 78% .

3. RNA-based Therapeutics RNA-based treatments like modified non-coding RNAs (ncRNAs), microRNAs (miRNAs), and others have been developed and studied by teams like Feng, Patil, et al (2021) to treat various diseases and conditions, including pancreatic cancer, acute renal failure, acute kidney injuries, diabetic macular edema, and advanced solid tumors.

In 2022, we expect to see further development of RNA-based therapeutics, like CAR T cells and other gene/cell therapeutics, therapeutic antibodies, and small molecular drugs to treat even more diseases and for prophylactic purposes as well.

4. Microvesicles + Extracellular Vesicles Microvesicles are coming to light due to their involvement in transporting mRNA, miRNA, and proteins — but how else might they support the human body? There are unknown functions of microvesicles and other extracellular vesicles that have yet to be discovered.

In 2020, Ratajczak and Ratajczak found that understanding microvesicles (or exosomes, microparticles) could mean understanding cell-to-cell communication , and their research showed that extracellular vesicles could transfer mRNA and proteins and modify stem cells ex vivo. This year, we look forward to seeing more research on what these tiny cell parts can do.

5. Metabolism in Cancers + Other Diseases Metabolism is the process of energy conversion in organisms and it represents the chemical reactions that sustain life. Recent research on metabolism in cancers and in immune cells has uncovered new ways to approach treatment and prevention of certain illnesses.

Take a look at Matsushita, Nakagawa, and Koike’s (2021) research on lipid metabolism in oncology and how recent advances in lipidomics technology and mass spectrometry have opened the door for new analysis of lipid profiles of certain cancers.

6. The Human Microbiome The human microbiome hosts bacteria, microorganisms, and other naturally-occurring flora that can help us and harm us. Diet, stress, drugs, and other factors shape the microbiome, leading to inflammation and an immune response of cytokines. Recent machine learning and statistical analyses of microbiome data , like that of Indias, Lahti, Nedyalkova, and team (2021) are getting smarter and smarter by removing variables and providing ways to test new hypotheses using statistical modeling.

With a deeper understanding of the microbiome, researchers like David Sinclair have shown that lifestyle changes can actually help people live healthier lives for longer . Sinclair’s lab is at the forefront of aging research and its impact on healthcare — and it’s all rooted in biotechnology and life science experiments.

Recent updates to ICD-11 and its classification of aging as a disease have led to debate, with Sinclair and colleagues advocating for the ongoing paradigm shift that biological age and chronological age are not synonymous. The implications for longevity and aging research from a funding perspective will be impacted by WHO and NIH decisions, and we anticipate seeing more biotech research on topics like epigenetics, metabolism, mitochondrial dysfunction, reproduction, and stem cell developments in the coming years.

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• Published: 11 May 2023

Applications of synthetic biology in medical and pharmaceutical fields

• Cuihuan Zhao 1 &
• Guo-Qiang Chen   ORCID: orcid.org/0000-0002-7226-1782 1 , 3 , 4  

Signal Transduction and Targeted Therapy volume  8 , Article number:  199 ( 2023 ) Cite this article

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• Biotechnology
• Nanobiotechnology

Synthetic biology aims to design or assemble existing bioparts or bio-components for useful bioproperties. During the past decades, progresses have been made to build delicate biocircuits, standardized biological building blocks and to develop various genomic/metabolic engineering tools and approaches. Medical and pharmaceutical demands have also pushed the development of synthetic biology, including integration of heterologous pathways into designer cells to efficiently produce medical agents, enhanced yields of natural products in cell growth media to equal or higher than that of the extracts from plants or fungi, constructions of novel genetic circuits for tumor targeting, controllable releases of therapeutic agents in response to specific biomarkers to fight diseases such as diabetes and cancers. Besides, new strategies are developed to treat complex immune diseases, infectious diseases and metabolic disorders that are hard to cure via traditional approaches. In general, synthetic biology brings new capabilities to medical and pharmaceutical researches. This review summarizes the timeline of synthetic biology developments, the past and present of synthetic biology for microbial productions of pharmaceutics, engineered cells equipped with synthetic DNA circuits for diagnosis and therapies, live and auto-assemblied biomaterials for medical treatments, cell-free synthetic biology in medical and pharmaceutical fields, and DNA engineering approaches with potentials for biomedical applications.

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Introduction.

The concept of synthetic biology was proposed in 1910s by Stephane Le Duc. 1 In this field, research strategies have been changed from the description and analysis of biological events to design and manipulate desired signal/metabolic routes, similar to the already defined organic synthesis. Unlike organic synthesis successfully developed in the early 19 th century, 2 synthetic biology is restricted by DNA, RNA and protein technology within the complexity of biological systems. Today, synthetic biology has been developed extensively. It becomes a multidisciplinary field aims to develop new biological parts, systems, or even individuals based on existing knowledge. Researchers can apply the engineering paradigm to produce predictable and robust systems with novel functionalities that do not exist in nature. Synthetic biology is tightly connected with many subjects including biotechnology, biomaterials and molecular biology, providing methodology and disciplines to these fields.

The timeline of synthetic biology developments is summarized here (Fig. 1 ). In general, the history of synthetic biology can be divided into three stages. The initial stage was found across the 20th century. Although the simplest organisms such as virus particles, bacteria, archaea and fungi were hard to engineer in the 20th century, some achievements were still acquired in the early explorations including the synthesis of crystalline bovine insulin, 3 chemical synthesis of DNA and RNA, 4 decoding of genetic codes 5 and the establishment of central dogma of molecular biology. 6 Synthetic biology has been accumulating its strengths in this period, as knowledge of genome biology and molecular biology are developed rapidly at the end of the 20th century (Fig. 1 ).

Timeline of major milestones in synthetic biology. The timeline begins at 1950s and expands to 2020s. Important events are listed in the right panels

The development stage begins in the 21 st century. In the first decade of the new millennium, synthetic biology is known to every biological researcher to include inventions of bioswitches, 7 gene circuits based on quorum sensing signals, 8 yeast cell-factory for amorphadiene synthesis 9 (Table 1 ), BioBrick standardized assembly 10 and the iGEM conferences 11 (Fig. 1 ). Two principles in synthetic biology designs have been considered in this stage including bottle-up 12 and top-down 13 ones, referring to the de novo creations of artificial lives by assembling basic biological molecules and engineering natural-existed cells to meet actual demands, respectively. However, most circuits are well-designed but still not enough for producing complex metabolites or sensing multiple signals, especially the applications are not well prepared for medical and pharmaceutical usages. Anyhow, synthetic biology is gradually becoming a most topical area, on the eve of rapid developments.

The fast-growing stage begins from the 2010s, the emergences of genome editing technologies especially CRISPR/Cas9, 14 low-cost DNA synthesis, 15 next-generation DNA sequencing 16 and high-throughput screening methods, 17 workflows of design-build-test-learn (DBTL) 18 and progresses in engineering biology 19 (Fig. 1 ), have allowed synthetic biology to enter a fast-growing period, 20 both in the lab-scale discoveries and industry-scale productions. Typically, Venter et al. assembled an artificial chromosome of Mycoplasma mycoides and transplanted it to M. capricolum to create new living cells. 21 Besides, new methods have accelerated the discovery and engineering of metabolite biosynthesis pathways, microbial artemisinic acid synthesis has been made possible, 22 , 23 which is the first industrialized plant metabolite produced by microbial cells. To realize the ultimate goal of design bio-systems similar to design electronic or mechanical systems, this is just the beginning. More efforts are needed to generate complex and stable biocircuits for various applications in the present of synthetic biology.

Besides scientists, investors also have realized the potentials in this field. Financial investments help establish synthetic-biology-related companies encouraged by the prediction that the global market of synthetic biology valued 9.5 billion dollars by 2021, including synthetic biology products (e.g., BioBrick parts, synthetic cells, biosynthesized chemicals) and enabling technologies (e.g., DNA synthesis, gene editing), 24 they are expected to reach 37 billion dollars by 2026. Most investments focus on medical applications. 25 Scientists and capital market are all optimistic about the future.

Started from chemical biosynthesis, synthetic biology has been expanded to cover areas in medical treatments, pharmaceutical developments, chemical engineering, food and agriculture, and environmental preservations. This paper focuses on the advances of synthetic biology in medical and pharmaceutical fields, including cell therapies, bacterial live diagnosis and therapeutics, production of therapeutic chemicals, nanotechnology and nanomaterial applications and targeted gene engineering.

Genetic engineering of therapeutic chassis

Engineered mammalian cells for medical applications.

With the advances in synthetic biology, researchers created various novel therapies using living cell chassis rationally designed from existing signaling networks with new constructs for their purposes, including e.g., production of medical biomolecules, synthetic gene networks for sensing or diagnostics, and programmable organisms, to handle mechanisms underlying disease and related organism/individual events (Fig. 2 ). We highlighted here synthetic biology strategies in mammalian cell engineering for metabolic disorders, tissue engineering and cancer treatments, as well as approaches in cell therapy and the design of gene circuits.

Development of smart living cells based on synthetic biology strategies. Smart cells can sense various environmental biomarkers, from chemicals to proteins. External signals are transducted into cells to trigger downstream responses. The products are also in the form of chemicals to proteins for customized demands. The sensing-reponsing system is endowing cells with new or enhanced abilities. P represents promoters

Therapies based on chimeric antigen receptor (CAR)-T cells

CARs are engineered receptors containing both antigen-binding and T cell-activating domains. T cells are acquired from patients and engineered ex vivo to express a specific CAR, and followed by transferring into the original donor patient, where they eliminate cancer cells that surface-displayed the target antigen. 26 CAR-T is a novel cell therapy began from 2000s. 27 The first generation of CARs are single-chain variable fragments (scFv) targeting CD19. 28 The development of artificial CARs comprises three generations. The first-generation CARs only contain a CD3ζ intracellular domain, while the second-generation CARs also possess a co-stimulatory domain, e.g ., 4-1BB or CD3ζ (Fig. 3 ). Studies with the third-generation chimeric antigen receptors with multiple co-stimulatory signaling domains are also under investigation (Fig. 3 ). 29 Because scFvs have the ability to recognize cell surface proteins, the targeting of tumors mediated via CAR-T cell is neither restricted nor dependent on antigen processing and presentation. CAR-T cells are therefore not limited to tumor escaping from MHC loss. For cancer immunotherapy, the main advantage of employing CAR-based methods is attributed to that the scFv derived from antibodies with affinities several orders of magnitude higher than conventional TCRs. 30 In addition, CARs can target glycolipids, abnormal glycosylated proteins and conformational variants that cannot be easily recognized by TCRs. Based on clinical trial results, there is an increasing evidence that CAR-T cells have the ability to deliver powerful anti-tumor therapeutic effects, leading to the recent FDA approval of CAR-T therapies directed against the CD19 protein for the treatment of acute lymphoblastic leukemia (ALL) and large B-cell lymphoma (DLBCL).

Synthetic biology in the designs of chimeric antigen receptors (CAR). a The AND gate used in artificial CARs. Three typical CARs i.e. Costimulation domain-based second-generation CAR, synNotch receptor-assisted CAR with multiple recognization mechanisms and chimeric costimulation receptor (CCR)-based CAR are exhibited from left to right. b The artificial CARs with inhibitory CAR (iCAR) system. The system can prevent recognizing self-antigens on somatic cells. c The artificial CARs sensing different tumor antigens. Two ScFvs recognizing different targets are tandemly fused, the engineered CAR can be triggered by multiple antigens. The figure is inspired by the paper 468

In addition, CAR-T applications are stepping into commercialization. The first approved CAR T-cell therapy was Kymriah which is CD19-targeted for treating DLBCL developed by Novartis and University of Pennsylvania. 31 DLBCL is a typical form of non-hodgkin lymphomas (NHL) that consist of 40% of total lymphomas. 32 The FDA also approved Yescarta (axicabtagene ciloleucel) in 2017 for DLBCL treatments. 33 In the clinical studies, patients with DLBCL were treated with the CD19-targeted CAR T-cells, with 25% partial responders and more than 50% complete responders. 34 , 35 Durable responses of over two years were observed, indicating the therapeutic effects of the CAR-T cells. However, cytokine storm, an excessive release of pro-inflammatory cytokines, was observed in Yescarta treated patients (13%), 36 indicating the safety needs to be improved.

The selection of target antigen is the determinant in CAR-T cell therapies. 37 , 38 , 39 If CAR-T cells can recognize protein expressed on non-malignant cells, severe cell toxicities could occur with the off-target activities. 40 The optimal target antigen is the one that is consistently expressed on the surface of cancer cells but not on the surface of normal cells. 37 , 41 , 42 Multiple myeloma is hard to treat via chemicals or stem cell transplantation. 43 , 44 CAR-T cell therapies are effective for multiple myeloma in preclinical studies. 45 However, to date, no antigen has been characterized that is strongly and constantly expressed on multiple myeloma cells but not on somatic cells. Among the antigens used so far, a member of the TNF superfamily proteins, B cell maturation antigen (BCMA), is the most favorable candidate for a multiple myeloma cell-directed CAR-T therapy target. 42 , 46 , 47 BCMA is expressed in cancer cells in almost all multiple myeloma patients, the expression of this antigen on somatic cells is limited to plasma cells and some kinds of B cells. 42 , 48 BCMA was the first antigen for multiple myeloma to be used in a clinical trial via a CAR-T cell approach leading to systematic responses in patients with this cancer. 40 , 42 , 49 Twelve patients received BCMA CAR-T cells in the dose-gradient clinical trial. Two patients treated with 9 × 10 6 CAR-T cells/kg body weight were obtained with good remissions, though the treatment had toxicity related to cytokine storms. 49 Many clinical trials investigating the safety and/or efficacy of anti-BCMA CAR-T cells are currently ongoing or finished.

Idecabtagene vicleucel (Abecma, also abbreviated as Ide-cel) is developed by Bristol-Myers Squibb, uses the anti-BCMA 11D5-3 scFv, the same as the 11D5-3-CD828Z CAR tested at the NCI. 49 However, the co-stimulatory domain is different, the CAR used in idecabtagene vicleucel is delivered using a lentivirus vector and has a 4-1BB co-stimulatory domain instead of a CD28 one. 50 In a multicenter phase I trial for idecabtagene vicleucel, 50 , 51 the therapy is highly effective for treating multiple myeloma patients. A phase II trial named KarMMa, designed to further evaluate the safety and ability of idecabtagene vicleucel, is undergoing. 52 The initial results of KarMMa demonstrates its deep, durable responses in heavily pretreated multiple myeloma patients. 52 Efficacy and safety were reflected in early reports, supporting a favorable idecabtagene vicleucel clinical benefit-risk profile across the target dose range in primary clinical results.

Receptor engineering in medical therapies

SynNotch receptors are a class of artificially engineered receptors that are used in medical applications (Fig. 3 ). 53 Notch receptors are transmembrane receptors participating in signal transductions, 54 comprising an extracellular domain, a transmembrane and an intracellular domain. 55 The transmembrane and intracellular domains are usually retained in synNotch architects, 56 whereas the signal-input extracellular domain is engineered to sense scFvs and nanobodies, 57 providing possibilities of recognizing agents to initiate signaling in living cells.

Also, the modular extracellular sensor architecture (MESA) was developed intending to detect extracellular free ligands 54 , 58 based on the synNotch idea. MESA designs have two membrane proteins each containing an extracellular ligand-binding domain which senses the chemicals or proteins and can be a small molecule-binding domain or antibody based sensing module, a transmembrane domain and either an intracellular transcriptional factor with relasing ability from the complex, protease recognition sequence or a protease. After ligand binding to the extracellular domain, MESA receptors dimerize and induce an intracellular proteolytic cleavage that allows the transcriptional factor dissociate for downstream regulations. The method allows more flexible sensor designs without limiting to Notch receptors. This system has also been remade recently to signal transduction via a split protease 59 or split transcriptional factor patterns. 60 The synNotch design has been constructed with a series of receptors called synthetic intramembrane proteolysis receptors (SNIPRs) containing domains from other natural receptors other than mouse Notch protein that are also cleavable by endogenous proteases. 61 Similiar to synNotch, SNIPRs bind to their antigens and function via dissociating a transcriptional factor to sense cell and immune factors. 62 For synNotch, SNIPR and MESA, the choice of ligand-binding domains and transcriptional factor domains enables customization of both sensing (signal input) and function (signal output) steps when using the systems. SNIPR and MESA also enrich the available engineering tools for the artificial receptor-effectors. However, some limitations still remain such as high background signals, off-target effects, the immunogenicity from the murine Notch protein, the large size of artificial receptors and transcriptional regulators. 56 , 61 , 63 Many efforts are needed to improve the system.

Receptor engineering applications are commonly related to CAR-T therapies. The receptors can be designed to target two specific antigens, one using the synNotch and the other via a traditional CAR. In preclinical models, T-cells engineered for targeting dual-antigen expressing cells are established. 64 TEV protease can be fused to MESA receptors, cleaving the transcriptional factor off for functionalization. 58 A humanized synthetic construct can reduce immunogenicity and minimize off-target effects. Zhu et al. constructed a framework for human SNIPRs with future applications in CAR-T therapies, preventing CAR-T cells from being activated via non-tumor signals. 61 Besides the above synthetic receptors, based on the same idea, Engelowski et al. designed a synthetic cytokine receptor sensing nanobodies by the fusion of GFP/mCherry nanobodies to native IL-23 intracellular domains. 65 Another receptor engineering strategy is to rewire receptor-transduced signals to novel effector genes. Using a scFv complementary to VEGF, the engineered receptor senses VEGF and released dCas9 protein, then the IL-2 expression are up-regulated. The system is successfully explored in Jurkat T cells. 58

The HEK-β cells used for diabetes treatments

β-cells are existing in pancreatic islets that synthesize and secrete insulin. 66 As the only site of insulin synthesis in mammals, β-cells sense blood glucose using a signal transduction pathway that comprises glycolysis and the stimulus-sensing-secretion coupling process. 67 , 68 The secretion of insulin is consisted with the following steps. Blood glucose is transported into β-cells and metabolized via glycolysis inside the cell, resulting in cell membrane depolarization, energy generation and closing of K + ATP channels, which activates the calcium channel Cav1.3 to induce calcium influx with the secretion of insulin granules. The excessive blood-glucose concentration in diabetes patients is from the deficiency of insulin-producing β cells for type 1 diabetes, or from low insulin sensitivity of body cells for type 2 diabetes. 69 Using a synthetic biology-based multiple screening approach, Xie et al. engineered human kidney cells HEK-293 to sense blood glucose levels for insulin secretion. 70 The design combines automatic diagnosis and treatment in diabetes therapy. The researchers demonstrated that overexpression of Cav1.3 provided the pathway for constructing a β-cell-like glucose-sensing module in somatic cells. 70 The combination of Cav1.3-controlled calcium and a synthetic Ca 2 + -inducible promoter allowed the monitoring of glucose levels using a tuned in vivo transcriptional response. After the construction of artificial HEK-293-β cells, the cell line HEK-293-β for glucose-response insulin production which maintained glucose homeostasis for over 3 weeks, via implanting the cells intraperitoneally to mice, also auto-corrected diabetic hyperglycemia within 3 days in T1D mice in this study.

The advantages of HEK-293-β cells are clear. Compared to primate pancreatic islets, HEK-293-β cells were adequately efficient in stabilizing postprandial glucose metabolism in T1D mice. Moreover, HEK-β cells are more easily for cultivation in vitro. It is expected that the engineered human cells have the prospect to be produced easily, cost-effectively and robustly, following current rules and regulations for pharmaceutical manufacturing, allowing the production of ready-to-use commercials with good properties for product purity, stability and quality. This highly innovative engineered cell raises the possibility that any cell type could be rationally reprogramming to achieve customized abilities such as blood glucose control.

The induced pluripotent stem cells (iPSCs) for medical applications

Synthetic biology also helps in generating human stem cells via overexpressing certain de-differentiation-related genes. One of the applications is the induced pluripotent stem cells. iPSCs are pluripotent stem cells generated from somatic cells. 71 Pioneered by Yamanaka’s lab, the introduction of four transcriptional factors including Oct3/4, Sox2, c-Myc, and Klf4, resulted in changing fibroblasts to embryonic stem (ES)-like cells, 72 which can re-differentiate into blood cells, bone cells or neurons for possible treatment of damages to various tissues and organs. 73 iPSCs are not created using human embryos, circumvented ethical concerns in contrast with ES cells. 74 Additionally, autologous somatic cell-derived iPSCs avoid immunological rejections. 75

iPSCs are self-renewable with continuous subculture properties. 76 The somatic cell samples from patients are induced into iPSCs able to serve as an unlimited repository for medical researches. The iPSC cell lines for Down’s syndrome and polycystic kidney disease are established. 77 , 78 An project termed StemBANCC calls for collections of iPSC cell lines for drug screening. 79 Various applications combined with therapeutic chemicals and iPSC cell lines are undergoing high-throughput drug screening and analysis. 80 , 81

iPSCs are aimed to be used for tissue regeneration and therapy developments. Type O red blood cells can be derived from iPSCs to meet demands for blood transfusion. 82 When cancer patients require large quantities of NK cells in immunotherapies, the cells can be manufactured using iPSCs to circumvent their low availabilities. 83 The anti-aging effects of iPSCs are observed during mouse studies. 84 The chemical-induced differentiation of iPSCs to cardiomyocytes has been commonly used. 85 These iPSC-cardiomyocytes are recapitulated with genetic codes in patients whom they derived, allowing the establishment of models of long QT syndrome and ischemic heart disease. 85 , 86 Cord-blood cells can be induced into pluripotent stem cells for treating malfunctional mice retina, 87 re-differentiated iPSCs are employed to cure brain lesions in mice with their motor abilities regained after the therapy. 88

iPSCs are successfully used for organ regeneration, for example, ex vivo cardiomyocytes can be used to regenerate fetal hearts to normal hearts via the Yamanaka’s method. 89 Human “liver buds” can be generated from three different cells including iPSCs, endothelial stem cells and mesenchymal stem cells. 90 The bio-mimicking processes made the liver buds self-packaging into a complex organ for transplanting into rodents. It functions well for metabolizing drugs. 91

Some iPSC applications are advanced to clinical stages. For example, a group in Osaka University made “myocardial sheets” from iPSCs, transplanted them into patients with severe heart failure, the clinical research plan was approved in Japan, 92 patients are under recruiting. Additionally, two men in China received iPSC-differentiated cardiomyocytes treatments. 93 They were reported to be in good condition although no detailed data are revealed. 93 iPSCs derived from skin cells from six patients are reprogrammed to retinal epithelial cells (RPCs) to replace degenerated RPCs in an ongoing phase I clinical trial. 94 Similarly, phase I clinical trials are also undergoing for thalassemia treatment using autologous iPSCs differentiated hematopoietic stem cells, 95 patients are recruiting. Till now, no Phase III study on stem cell-related therapy has been conducted. The major concern is the safety of iPSCs with the carcinogenic possibilities: teratoma has been observed in iPSCs injected mice, 96 low-induction efficiency, incomplete reprogramming of genomes, immunogenicity and vector genomic integrations are also issues of concerns. 97 , 98 More efforts are required for clinical applications.

Synthetic biology in tissue engineering

Tissue engineering aims to repair damaged tissues and restoring their normal functions. The use of synthetic biology in tissue engineering allows control of cell behaviors. Artificial genetic constructs can regulate cell functions by rewiring cellular signals. As engineered cells are building blocks in tissues with special properties to achieve smarter functions, synthetic biology allows complex tissue engineering for new medical studies.

By overexpression of functional genes or transcriptional factors, stem cells can differentiate to generate specific tissue cells successfully. 99 This is a simple and common way in stem cell-based tissue engineering. However, the gene overexpression lacks feedback control mechanisms to avoid excess nutrient consumption or cell toxicity. 100 For an instance, constitutive overexpression of the anti-apoptotic factor Bcl-2 leads to tumorigenesis risks. 101 , 102 CRISPR/dCas9 bioswitches or synthetic mRNAs are found able to solve the problem via time and spatial-specific expression of genes. 103 , 104 Moreover, introductions of genetic circuits sensing small molecules or cell-surface proteins are well studied, especially Tet repressor-based system. 105 Gersbach et al. designed a Tet-off system controlling Runx2 factors that can regulate the in vivo osteogenic processes. 106 Yao et al. employed a Tet-on system to express Sox9 specifically in engineered rat chondrocytes, Sox9 is a key factor maintaining chondrocyte viability, activating the protein expressions for type II collagen and aggrecan in cartilage tissue engineering. 107 Chondrocyte degradation was inhibited after Dox (Tet system inducer) injection in implanted cell scaffolds. 107 The Tet-on system is also used for overexpressing interleukin-1 receptor antagonist (IL-1Ra) gene to modulate inflammatory cytokines during the chondrogenesis processes in cartilage repairs 108 (Table 2 ). Tet-switches have aided elapsed time controllable gene expressions for tissue engineering.

The optogenetic induction systems are also used in the control of cell behaviors in tissue engineering. Light inducible proteins are able to respond to UV and far-infrared lights, making light induction applicable. 109 Various optogenetic circuits are constructed by fusing light-sensitive motifs to well-characterized transcriptional factors. 110 , 111 Spatial-specific gene activation has been successfully employed to guide the arrangement of cells. 112 Sakar et al. used blue light-induced channel rhodopsin-2 to achieve dynamic and region-specific contractions of tissues. 113 The optogenetic control of engineered murine-derived muscle cells offers remote gene activation or silencing via the light-sensitive membrane Na + channel and ion-inducible downstream elements for tissue engineering.

Inspired by successes of CAR-T cells, G protein-coupled receptors (GPCRs) are engineered to sense artificial ligands for tissue engineering. 114 Park et al. successfully designed and used a GPCR sensing clozapine-N-oxide (CNO) in primary cells for the control of cell migration in response to CNO concentration gradients. 115 This technology could make a valuable module for wound healing and cell regeneration. Synthetic biology makes possible to program cells to multicellular structures in a self-assembly manner. 116 Toda et al. employed synNotch methods to engineer cell adhesion signals in a population of mouse fibroblasts that were turned into multilayers and polarized according to the synNotch receptor types. 117

Besides cells, biomaterials are commonly used in tissue engineering, served as scaffolds and bio-mimicked organs. 118 the auto-modulation characteristics of biomaterials in response to stimuli or chemical compounds are useful in biomaterial-based tissue engineering. Baraniak et al. engineered the B16 cell line with a green fluorescent protein (GFP) reporter induced by RheoSwitch Ligand 1 (RSL1), which was coated on poly(ester urethane) films, allowing GFP activation for up to 300 days on the film. 119 Deans et al. constructed an isopropyl-β-d-thiogalactoside (IPTG)-induced Lac-off system in Chinese hamster ovary (CHO) cells, and IPTG encapsulated in poly(lactide- co -glycolide) (PLGA) scaffolds or PEG beads was released in a sustainable manner. The reporter gene indicated that the induction lasted over 10 days in mouse models implanted subcutaneously into the dorsal region, 120 the GFP fluorescence level was observed to be controlled by its locations. 121 The spatial-induced gene expression regulation has become a design-of-concept in many applications like cartilage repair and in vivo 3D cell scaffolds.

In summary, expressions of biological circuits could generate functionalized cells for tissue engineering. Multiple synthetic biology designs e.g . time and spatial-dependent gene expression, induction and autoregulation systems and smart biomaterials are available in this field. The state-of-the-art development still remains with many obstacles from moving truly synthetic tissues into clinic, but at least some foundations are settled for future studies.

Engineered bacterial cells for therapeutical applications

Synthetic biology approaches have promoted genetically engineered bacteria for novel live therapeutics (Fig. 2 ). 122 Bacteria containing synthetic gene circuits can control the timing, localization and dosages of bacterial therapeutic activities sensing specific disease biomarkers and thus develop a powerful new method against diseases. 123 Synthetic biology-based engineering methods allow to program living bacterial cells with unique therapeutic functions, offering flexibility, sustainability and predictability, providing novel designs and toolkits to conventional therapies. 124 Here some advances are presented for engineered bacterial cells harboring gene circuits capable of sensing and transduction of signals derived from intracellular or extracellular biomarkers, also the treatments and diagnosis based on these signaling pathways. The concept of bacterial cell-based live therapeutics and diagnostics are rapidly growing strategies with promises for effective treatments of a wide variety of human diseases.

Engineered bacterial cells in cancer diagnosis and treatments

Some anaerobic/facultative anaerobic bacterial cells are good candidates for tumor treatments. They can target the anaerobic microenvironment of tumors, they also have the tumor lysis-inducing and trigger inflammation abilities useful in fighting against solid tumors. 125 Engineered microbes can become suitable tools for cancer in vivo diagnosis. Danino et al. engineered E. coli with LacZ reporter gene, the bacterium produces LacZ when in contact with tumor cells. Subsequently, mice were injected with chemiluminescence substrates for LacZ (Table 3 ). The luminescence is enriched in the urine to generate red color. 126 The method is more sensitive than microscopes as it can detect tumors smaller than 1 cm. Similarly, Royo et al. constructed a salicylic acid-induced circuit converting 5-fluorocytosine to toxic products in attenuated Salmonella enterica for tumor killing. 127 Salmonella enterica localized in tumor tissues after the injection, with the additional providing of salicylic acid (inducer) and 5-fluorocytosine (substrate), tumor cells were eliminated via the formation of 5-fluorouracil from the bacterial cells.

To improve the effects of bacteria-based cancer therapies, some studies aim to further enhance bacterial tumor tropism. 128 Some bacteria have natural affinity for the anaerobic environment of solid tumors, like E. coli or attenuated Vibrio cholerae , Salmonella typhimurium , and Listeria monocytogenes . 128 However, the affinity is not sufficient for targeted therapies, bacterial cells in vivo are still dispersed in general. They can be augmented by introducing synthetic surface adhesins targeted to bind cancer-specific molecules like neoantigens or other chemicals or proteins that are enriched in cancer cells, not accumulated in somatic cells. Engineering of adhesins are demonstrated to be effective in enhancing bacterial tumor reactions. The adhesins are membrane-displayed proteins with extracellular immunoglobulin domains that can be engineered via library directed evolution screens. Piñero-Lambea et al. constructed a constitutive genetic circuit in E. coli with an artificial adhesin targeting green fluorescent protein (GFP) as the evidence of a proof of concept, it demonstrated the abilities from that binding of the cell membrane-engineered bacteria to GFP-expressing HeLa cells are successful both in vitro and in mice. 129 Importantly, the intravenous delivery of this engineered bacteria to mice resulted in effective and efficient colonization in xenografted solid tumors of HeLa cells at a dose 100 times lower than that for a bacterial strain expressing an irrelevant control adhesin, or for the wild-type strain, suggesting that similarly engineered bacteria can be used to carry therapeutic agents to tumors at low doses with marginal potential systemic basal toxicities. 130 , 131 However, few tumor-targeting bacteria have entered clinical stages. The facultative anaerobe Salmonella typhimurium VNP2000, has been engineered for safety with anti-tumor abilities in pre-clinical studies, 132 yet it failed in the phase I clinical trial for marginal anti-tumor effects and dose-dependent side effects. 133 Some other clinical investigations based on bacteria Clostridia novyi -NT or Bifidobacterium longum APS001F are ongoing or recruited for their phase I trials. 134

Engineered bacterial cells for diabetes diagnosis and treatments

Bacteria have been engineered to detect glucose concentrations for diabetes. Courbet et al. described an approach in sensing abnormal glucose concentrations in human urine samples. 135 They encapsulated the bacterial sensors in hydrogel beads, glucose in urine will change the color to red in beads. The in vitro bacterial glucometer has found outperforming the detection limit of urinary dipsticks by one order of magnitude.

Some proteins and peptides are biosynthesized in engineered gut bacteria for diabetes treatments. The engineered probiotic L. gasseri ATCC 33323 produced GLP-1 protein, the bacterium is orally delivered to diabetic rats, 136 demonstrating a down-regulation of blood glucose levels to 33%. Similarly, engineered L. lactis FI5876 was reconstructed to biosynthesize and deliver incretin hormone GLP-1 to stimulate β-cell insulin secretion under conditions of high glucose concentrations. Results showed the glucose tolerance is improved in high-fat diet mice. 137 The probiotic L. paracasei ATCC 27092 is engineered to secret angiotensin (1-7) [Ang-(1-7)], increasing the concentrations of Ang-(1-7) (an anti-inflammatory, vasodilator and angiogenic peptide phamarceutical), and reduced the side effects on retina and kidney in diabetic mice, as the insulin production level is increased after oral administration of the bacteria. Following the design, oral uptake of engineered B. longum HB15 which produces penetratin (a cell-penetrating peptide with the ability of enhancing delivery of insulin), and GLP-1 fusion protein also enhanced the production of GLP-1 in the colorectal tract. 138 , 139 , 140 L. paracasei BL23 was also successfully designed to produce monomer GLP-1 analogs displayed to the bacterial membrane via fusing GLP-1 to peptidoglycan-anchor protein PrtP, the engineered bacteria enhanced glycemic control in rats with diabetes. However, the efficacy is still limited and needed further investigations. 141 In addition to GLP-1, some other proteins like the immunomodulatory cytokine IL-10 along with human proinsulin were simultaneously introduced to engineered L. lactis MG1363, the combination therapy with low-dose systemic anti-CD3 allowing reversal of irregulated self-autoimmune triggered diabetes in non-obese diabetic mice. 142 , 143 This design could possibly be effective for the treating of type 1 diabetes in human.

Engineered bacterial cells for diagnosis and treatments of gastrointestinal diseases

Probiotics can be used to treat inflammatory bowel disease (IBD). 144 IBD is chronic inflammation of tissues in the digestive tract, including ulcerative colitis and Crohn’s disease. Patients are suffering from diarrhea, pain and weight loss. Synthetic biology approaches and ideas help bacteria acquire more powerful abilities against gastrointestinal diseases. Praveschotinunt et al. designed an engineered E. coli Nissle 1917 (EcN) that produces extracellular fibrous matrices to enhance gut mucosal healing abilities for alleviating IBD in mice. 145 Curli fibrous proteins (CsgA) were fused with trefoil factor (TFF) domains to promote the reconstruction of cell surface, and the bacterium could produce fibrous matrices via the in situ protein self-assembly of the modified curli fibers. The results revealed that the designed EcN significantly inhibited the production of pro-inflammatory cytokines, alleviated the weight loss of mice, maintained colon length, demonstrating its anti-inflammation ability in the dextran sodium sulfate (DSS)-induced acute colitis mouse model. The design could be expanded to a general approach for probiotic-based live therapeutics in IBD treatments.

Bacteria are feasible to be engineered to directly eliminate pathogens for preventing infectious diseases in gastrointestinal tracts. Pseudomonas aeruginosa is a common multidrug-resistant pathogen difficult to treat. Engineered EcN has been employed for the detection, prevention and treatment of gut infections by P. aeruginosa . 146 The designed EcN was able to sense the biomarker N-acyl homoserine lactone produced by P. aeruginosa , and autolyzed to release a biofilm degradation enzyme dispersin and pyocin S5 bacteriocin to eliminate the pathogen in the intestine. Moreover, the reprogrammed bacteria displayed long-term (over 15 days) prophylactic abilities against P. aeruginosa and was demonstrated to be more useful than treating a pre-established infection in mouse models. 3-Hydroxybutyrate (3HB) is a component of human ketone bodies with therapeutic effects in colitis. Yan et al. constructed an EcN overexpressing 3HB biosynthesis pathway. 147 Compared to wild-type EcN, the engineered E. coli demonstrated better effects on mouse weights, colon lengths, occult blood levels, gut tissue myeloperoxidase activity and proinflammatory cytokine concentrations. 147 However, the studies are the preliminary results in mice, they have not reached clinical trials yet. Further efforts are needed to evaluate their applications in human.

Engineered bacterial cells for metabolic disorders

Engineered gut microbes also have been used to target metabolic disorders. 148 E. coli was designed to treat obesity synthesizing anorexigenic lipids precursors in mice with high-fat diet. 149 Some efforts are made to degrade toxic compounds accumulated in patients via live bacteria. Kurtz et al. engineered an E. coli Nissle 1917 strain for converting ammonia to L-arginine in the intestine and reducing systemic hyperammonemia in both mouse and monkey models. 150 Isabella et al. reprogrammed E. coli Nissle 1917 to overexpress phenylalanine degradation pathway to metabolize excess phenylalanine in phenylketonuria (PKU) patients. In the Pah enu2/enu2 PKU mouse model, oral uptake of the engineered bacterium significantly down-regulated blood phenylalanine concentration by 38%. 151

Alcoholic liver disease is the major cause of liver disorders, widely risking the health of heavy drinkers. 152 The engineered Bacillus subtilis and L. lactis could be employed to express ethanol degradation pathway (alcohol dehydrogenase and aldehyde dehydrogenase) for the detoxification of alcohol and alleviate liver injury from alcohol overconsumption. 153 Moreover, the lectin regenerating islet-derived 3 gamma (REG3G) protein is decreased in the gastrointestinal tract during chronic ethanol uptake. L. reuteri was designed to overexpress the interleukin-22 (IL-22) gene, which increased REG3G abundance in the intestine, reduced inflammation and damage in liver using an alcoholic liver disease mouse model. 154

Synthetic biology approaches have allowed the construction and design of engineered live biotherapeutics. Many cases are targeting future clinical applications. The examples discussed here indicate that, with the development of circuit designs and understanding in microorganism hosts, researchers can construct live biotherapeutics that function in a precise, systematic, inducible and robust manner. However, many efforts are still needed to weaken bacterial toxicity and increase the controllability in vivo.

Synthetic biology in the fabrication of emerging therapeutic materials

Besides engineered cells, engineered nanomaterials are also commonly used in medical fields. Nanobiotechnology aims to solve important biological concerns similar to drug delivery, disease diagnosis and treatment based on its unique physical, chemical and biological properties of micro-nano scale materials 155 , 156 (Fig. 4 ). Nanomaterials possess unique mechanical, magnetic and electronic properties, able to respond to external signals, controlling their downstream circuits. 157 However, traditional nanomaterials are generated from physical and chemical processes, the solvents and modifying molecules are frequently causing bio-safety issues. 158 Recently, biological nanomaterials have been developed exhibiting their advantages in environmentally friendly, enhanced biocompatibility and bioactivity, and low tissue toxicity under the guidance of synthetic biology. 159 Based on synthetic biology concepts and approaches, the genetic engineered bacteria, 160 yeast 161 and tobacco mosaic virus 162 (TMV) can serve as bio-factories for nanomaterials. 163 Mammalian cell-derived vesicles and nanoparticles have suitable biocompatibility, also commonly used as nanomedicines. 164 Biological materials can be constructed and engineered with the help of synthetic biology, extending their application scenarios in modern disease treatments.

The designs and applications in synthetic material biology. Generally, a genetic circuit is constructed to synthesize biological materials or sense environments. The engineered bacteria are endowed with new characteristics like color change and unique surface properties. The applications for cells with excellular matrices are diverse including magnet field induced therapies, development of novel drug carrier or health monitoring via sophiscated biofabrication processes. This figure is partially inspired by the paper 469

Synthetic biology in the artificial organelles

Following the principles of synthetic biology, biocatalysis or trigger-sensing modulus nanoparticles can be processed to self-assembly organelles, 165 , 166 which are biomimicry of characteristics of living cells like enzyme reaction compartmentalization and stimuli-responses (Fig. 4 ). The design also provides new inputs for constructing artificial cells. 167 Additionally, combinations of artificial organelles and engineered living cell chassis including CAR-T cells and engineered bacteria, the nano-living hybrid system can exert its dual effects to enhance therapeutic results or more strictly control of artificial systems.

Polymersomes are artificial hollow vesicles made by amphiphilic polymers, using as shells of artificial organelles. van Oppen et al. employed a polymersome-based system that was anchored with cell-penetrating peptides on its outer membrane. The artificial organelles possess inside catalase, allowing degradation of external reactive oxidative molecules, perform as a synthetic organelle, protecting the cells from ROS damages triggered via H 2 O 2 , which showed abilities in uptaking by human primary fibroblasts and human embryonic kidney cells. 168 A similar design relying on polymersomes equipped with two enzymes and related transmembrane channels, was used to mimic cell peroxisomes. These organelles were able to deal with both H 2 O 2 and superoxide radicals. The results further demonstrated the feasibility of artificial organelle with catalase activity. Based on similar ideas, engineered polymersomes may play a role in treating medical conditions including Parkinson’s, Alzheimer’s, Huntington’s, metabolic diseases, cancers and acatalasemia via harboring various therapeutic proteins inside of the artificial organelles. 169 , 170

Moreover, the fusion of nanobiotechnology and synthetic biology may achieve novel functions. First, researchers can create “artificial lives” via assembling nanoparticles following the “bottle-up” principle. The idea can be applied in constructing biological components using inorganic scaffolds and functional nanomaterials with nucleic acids and protein inside of the nanoparticles. 171 , 172 The “top-down” principle, or engineering natural cells for actual demands, can be used as a guidance when using nanomaterials in living cells for chimeric biological systems to increase the robustness, stability and sensitivity in specific medical applications.

Constructing nanoparticle-mediated genetic circuits

Auto-responses can be achieved via internal environmental stimulus to induce genetic switch ON/OFF 173 (Fig. 4 ). However, the irreversible situation of genetic switches is a common and difficult problem. 174 , 175 To circumvent the weakness of genetic constructs, nanoparticles are employed to sense signals for the transductions in vivo. Light, sound, heat and magnet stimuli are easy to respond for nanoparticles, they can be used as inducer systems for solid tumor and diabetes treatments. Yet the spatial-specific induction is hard for physical stimulus. 176 Overall, via combining the advantages of genetic sensor and nanoparticles, it is feasible to convert physical stimuli into genetic switch with specified input signals by introducing nanoparticles for signal transduction, and the time-spatial control of gene expressions are realized. 177

Near-infrared (NIR) light-responsible gene circuits are feasible for in vivo therapeutical applications for their better transmission of NIR light able to penetrate tissues and lower toxicity. 178 NIR-sensing protein is identified in plants and bacteria, like the bacterial phytochromes (BphPs). 179 However, NIR-sensing proteins are generally with low brightness. 180 Also, the lacking of structural information hindered their rational engineering. 180 To circumvent the disadvantages of NIR light-responsible protein, researchers have use nanomaterials converting NIR light into visible light. For example, Chen et al. employed nanoparticles doped with lanthanide to derive 980 nm NIR light into visible light, controlling genetic gates of opsin-expressing neurons in mice models. 181 , 182 Another design uses plasmonic gold nanorods or photothermal responsible nanoparticles to transduct NIR light into up-regulation of temperature, then the promoters of heat-shock protein are activated for downstream gene expression. 183 , 184 One disadvantage for nanoparticles is that they must be injected into human body, it could be solved by developing genetically engineered nanoparticles. 185 Similar to magnetogenetics, in which biosynthesized ferritin can be used as a tool to prepare exogenous paramagnetic nanoparticles. However, the penetration depth needs much improvements in these samples (less than 1 cm), which is not enough for the applications of cell therapy demands in humans. Some researchers couple light-generating microdevices with photosensitive engineered therapeutic cells to address the problem (Fig. 4 ), 186 , 187 , 188 patients can control the release of drugs via applications of their own smartphone or real-time monitoring their health. Besides, some genetic-encoded luminescent module can produce light in situ with a protein like various luciferases, all emit the desired wavelength with corresponding substrates. The in vivo light induces the photosensitive proteins that trigger transgene expressions for customized demands. 189

In addition to optogenetics, magnetogenetics emerges for regulating the cell activities and has been applied for controlling of nanomaterial therapies remotely and non-invasively (Fig. 4 ). 190 , 191 , 192 , 193 Magnetic fields can penetrate human body without losses, which is a preferred characteristic in deep-tissue targeted therapies. Previous magnetogenetics tools are mainly externally injected magnetic nanoparticles. 190 , 192 , 194 The nanoparticles are usually with radius of <10 nm, toxicity free and water-soluble. 190 Heating of nanoparticles using remote magnetic fields can activate temperature-sensitive cation channels in cells. the next-generation tools are heterologously expressed receptor-targeted ferritin proteins in the form of nanoparticles (iron-loaded particles) in engineered cells, which could sense and transduce magnetic signals to cell membrane-anchored receptors like transient receptor potential channel 1 (TRPV1) or TRPV4. 191 , 193 The membrane receptors are ion channels allowing calcium influx with the magnet stimuli. The described gene circuit can be manipulated to control NFAT-dependent transcriptional regulators for downstream functional genes. Implanted engineered therapeutic cells can achieve target-specific treatments and precise control of therapeutic dosage, time and location under magnetic fields.

However, the mechanisms of the magnetic activation of the sensor channels are still not clear, the theories proposed are under debate for a long time. 195 TRPV channels are activated by a variety of signals including but not limited to mechanical forces and heat. Recently, a new mechanism is raised to solve the problem that how radio-frequency weak magnetic fields (1 mT) could trigger transient responses in living cells with ferritin-anchored TRPV channels. 196 The mechanism is the dissociation of free Fe 3+ from ferritin protein, resulting in an enhanced oxidation of membrane lipids via increased production of reactive oxygen species (ROS). 196 These oxidized lipids have the ability to turn on the TRPV channels, resulting in calcium influx. 196 , 197 , 198 Recently, ROS is reported to be involved in the treatment of combined electric and static magnetic fields in type 2 diabetic mice to increase their insulin sensitivity. 199 In this research, low-energy fields can induce the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator controlling ROS levels. 199 Moreover, the local ROS accumulation does not have side effects in mice, it is promising to induce gene expression via electromagnetic fields mediated by redox states. 200 Magnetogenetics are exhibiting its potentials in remote control and targeted therapies. However, more efforts are needed to establish the magnetogenetic platform. Despite improvements in recent years, the cell toxicity and biocompatibility are two main obstacles of magnetic nanoparticles that still challenges their in vivo applications.

Synthetic biology in drug delivery

The synthetic biology constructs are usually encapsulated in carriers for their functions in vivo. The safety concerns of viral vectors restrict their applications for editing human genome. 201 Therefore, non-viral carriers are attracting more and more attentions. Nanotechnology can aid to deliver therapeutic agents including genetic circuits and genome engineering tools. 202 , 203 With the advances in nanotechnology, more choices are available for targeted and controllable-release in DNA/RNA delivery system. 204

One of the examples, the DNA/RNA delivery system based on liposome nanomaterials, has become an effective and potential gene therapy method, with a variety of artificial lipid vectors approved for clinical uses. For example, an RNAi therapeutic agent under the trade name Onpattro, has been developed by Alnylam Pharmaceuticals. The drug was approved in 2018 for the treatment of polyneuropathy. 205 Liposomes are small lipid vesicles, the size is between 50 nm and 1 μm. 206 Liposome are generally amphiphilic consisted with a hydrophobic tail and a hydrophilic head, employed for delivering drugs in various treatments. 207 Because liposomes reduce drug toxicity, deliver drugs directly to targets via site-specific injections, and envelope drugs free from degradation, they have advantages over traditional drug therapies in delivery. CRISPR/Cas9-aided gene therapies are commonly using lipid-based nanoparticles integrating negatively charged mRNA, gRNA scaffolds and CRISPR genes with positively charged liposomes via electrostatic interactions. 208 Felgner et al. first designed and used liposomes by enveloping DNA and delivered it to target mammalian cells in the plasma membrane, leading to DNA expression after its endocytosis. 209 The liposome vector not only helps therapeutic DNAs to pass through the cell membrane barrier, but also protects them from DNase degradation and immune responses to maintain their activities. Partially inspired by the results that liposomes can be applied in human therapies, liposomes also have delivered mRNA encoding SARS-CoV-2 antigens to humans as vaccines. Both the Moderna mRNA-1273 and BioNTech/Pfizer BNT162b2 vaccines are encapsulated in liposomes, with their clinical use approvals. 210

Nanotechnology can also aid synthetic biology to deliver chemicals. 211 , 212 Nanocarriers deliver chemicals minimize off-target effects, 213 , 214 enhancing therapeutic results 215 , 216 compared to traditional drug administrations. External physical stimuli can also initiate the release of chemicals to make the system sustainable and controllable. 217 Here, we discuss the application of synthetic biology-guided biological chemical carriers.

The genetically encoded post-translational modified protein can self-assemble to carry hydrophobic drugs. 218 The protein with different structure and material properties can be easily manipulated at the amino sequence level. Based on synthetic biology approaches, Mozhdehi et al. designed and co-expressed an elastin-like polypeptide and an N-myristoyl transferase in E. coli . 219 The N-myristoyl transferase enzyme modified the polypeptide with myristoyl groups in bacteria, generating a temperature-induced self-assembly behavior. 219 The lipid core of the purified recombinant protein can carry hydrophobic compounds with a prolonged drug half-life. 220 The protein can form complex assembly systems encapsulated with chemicals. Li et al. used an in silico designed cationic chimera near-infrared fluorescent protein and anionic carboxylate-terminated PEG to prepare a protein-PEG nanocarrier. 221 The nanoprotein is amphiphilic, resulting in the aggregation and phase separation in aqueous solutions to form nanoparticles. 221 The engineered nanoparticle achieved imaging of solid tumor and metastasis in vivo without transfections for the fluorescent nature of the protein, 221 as well as the nanoprotein served as the long-term drug carrier, which can improve half-life and therapeutic effects of IL1-Ra significantly. 222

Engineered bacterial outer-membrane vesicles (OMVs) as nanocarriers

Bacterial outer membrane vesicles (OMVs) are lipid spheres released from Gram-negative bacterial outer membranes, they can be used for trafficking biochemicals to other cells in the environment. 223 The gene manipulation methods from synthetic biology can improve bio-originated nanoparticle abilities, 224 expanding the application scenarios of outer-membrane vesicles (OMV) and engineered cells. 225 , 226

Engineered OMV anchored with recombinant proteins are potentially used in medical and clinical fields (Fig. 4 ). The general strategy to surface display proteins in the engineering of OMV is to fuse their genes together in the OMV expression system. Many studies have employed the E. coli Cytolysin A (ClyA) protein as the fusion chassis to anchor exogenous proteins to OMV membranes. 227 , 228 , 229 , 230 In recent studies, ClyA has been reported to successfully fuse to the domain 4 of Bacillus anthracis protective antigen, to extracellular domain of the influenza A matrix protein 2 (M2), and to GFP without influences OMV formation. 231 The alternative strategy is to express proteins to the periplasm and assembly to the OMV when the fusion step hampers protein functions. 232 However, the heterologous protein is enveloped inside of the OMV, which is a main disadvantage of the strategy. Bartolini et al. also employed the method to carry Chlamydia muridarum protein HtrA in OMVs as a vaccine against Chlamydia infections. 233 , 234 Some proteins from Streptococcus spp. are expressed to the periplasm with the E. coli OmpA signal peptide to packed them into OMVs. 235 Even though these proteins are located inside of the OMV, they were able to activate the immune responses, 232 , 233 , 235 the generated IgG antibodies had strong activity to specific pathogens in murine models. 225 , 232 , 235 The results indicated that antigen location is not a decisive factor in OMV-elicited immune responses.

Besides proteins, OMVs can be engineered to carry chemicals. LPS and capsular polysaccharides (CPS) decorating the cell membrane of pathogens are also vaccine candidates. 236 However, polysaccharides trigger immune responses apart from T-cells, the immunological memory cannot be established. 237 To circumvent the problem, polysaccharides are anchored to nanocarriers to elicit immunological memories. Polysaccharide and capsule synthesis genes are expressed in E. coli , packed into OMVs using the mentioned methods. The designed OMVs are potentially used as vaccines after further optimizations. Chen et al. employed the O-antigen polysaccharide from Francisella tularensis , the genes were heterologous expressed in E. coli to produce the glyco-modified OMVs. 238 , 239 Mice injected with the engineered OMVs were protected against F. tularensis strains. 238 Another similar design uses Streptococcus pneumoniae CPS (Sp-CPS) biosynthesis genes. They were overexpressed in E. coli , located both on the membrane of engineered OMVs and bacterial cells. 240 , 241 After the vaccination via injecting these collected OMVs, the vaccine was effective in opsonophagocytosis assays and IgG antibodies were triggered against Sp-CPS. 240 In general, synthetic biology approaches have developed better engineered OMVs for immunotherapies, 242 , 243 with bright prospects in drug targeted-delivery and combined therapies.

Biomimetic medical adhesive materials

Traditional medical adhesive materials are limited in underwater uses, which hampered their applications in body fluids. Recently, some biomimetic designs are conducted to solve the problem based on synthetic biology ideas (Fig. 4 ). 244 Many marine organisms (e.g. mussel and barnacle) have extraordinary adhesive capacities to rock surfaces, 245 , 246 as they produce L-3,4-dihydroxyphenylalanine (DOPA) as an important component of the adhesion proteins in underwater surfaces. 247 Zhong et al. reported a strong underwater adhesive by fusion of CsgA curli protein and mussel foot proteins. 248 The excellent design reconciled the biocompatibility and adhesion activity, with the prospect of in vivo applications like tissue repairs. Zhang et al. is inspired by natural biomaterials like bones and mussel foots, 249 they developed a Bacillus spp. extracellular matrix-based living glue. 250 The live material is adhesive with regeneration abilities. Engineered mammalian cells could be constructed with adhesive proteins, serving as in vivo live functional glues. As summarized above, the novel live biomedical adhesives are hotspots in medical synthetic biology. However, most studies are focused in the material properties rather than their biocompatibility and biodegradability, adequate efforts are needed to promote the material for clinical applications.

Genetically encoded click chemistry in medical applications

Inspired by click chemistry, isopeptide bond was engineered for the establishment of protein-protein linkages. 251 The genetic-encoded click chemistry is more applicable in living organisms compared with traditional click chemistry. The SpyTag/SpyCatcher system is an application of the natural click-like reaction among Gram-positive bacterial pilus, 252 , 253 using biological ways to form stable chemical bonds between amino acids, additional modifications of biomacromolecules are not needed in click chemistry-oriented proteins (Fig. 4 ). 254 Genetically encoded click chemistry (or Spy chemistry) is a powerful tool for materials made via synthetic biology. 255

Hydrogels are cross-linked hydrophilic polymer networks, 256 serving as carriers for biomacromolecules and stem cells due to their biocompatibilities and extracellular matrix (ECM) like properties. 257 Hydrogel materials synthesized using chemical polymerizations are facing bioactivity problems. 258 The protein characteristics are decided by amino acid sequences. Protein hydrogels are easier to synthesize and be controlled using various DNA sequences. Yang et al. employs the SpyTag/SpyCatcher system to synthesize a 4-arm star-like light-sensing protein. The protein can form rapid sol-gel and gel-sol phase transitions in response to AdoB 12 and light, respectively. 259 Biofilm-degrading glycosyl hydrolase PslG can be enveloped into the hydrogel, endowing the material with abilities against multidrug-resistant bacteria in chronic infections. Sun et al. designed a Spy-network containing multiple SpyTags and SpyCatchers in elastin-like proteins and the leukemia inhibitory factor. The proteins were turned into a high-mechanical strength hydrogel, allowing mouse embryonic stem cells to maintain pluri-potentials without adding other cytokines in the gel. 260

Genetically encoded click chemistry has also used in the vaccine development. Some designed proteins can self-assembly into virus-like particles (VLPs) to surface display antigens for mimicking pathogens. 261 Synthetic vaccines are causing more and more attentions for their efficiency and safety compared to canonical vaccines developed from dead or attenuated microorganisms. Genetically encoded click chemistry is a useful approach to modify the surface with heterologous antigens to enhance their immunogenicity. 262 , 263 The easy formation of chemical bonds based on Spy chemistry provide a customized and convenient method to design synthetic vaccines via encoded protein self-assembly. Liu et al. developed a synthetic vaccine using the SpyCatcher/SpyTag chemistry via covalently ligating specific antigens and chemicals. The result demonstrates this engineered vaccine targets dendritic cells successfully. 264 The generated protein-chemical hybrid vaccine remained the individual functions and had the ability to trigger B and T cell responses. Brune et al. engineered virus-like particles (VLPs) via exhibiting SpyCatcher on material surfaces, further enabling the modification of VLPs with SpyTag-expressing malarial antigens to develop novel vaccines. 265 The VLP-antigen vaccine can trigger immune responses rapidly and efficiently via only one single immunization, indicating the potential of this effective, simple, and modular modification method.

Genetic code expansion for medical and pharmaceutical applications

A protein usually consists of 20 natural amino acids. To add non-canonical amino acids (ncAAs) into proteins, the genetic code expansion technology has been developed. 266 ncAAs can be used to modify proteins via conjugation with peptides or chemicals depending on actual demands. Employing a termination codon (UAG/UGA/UAA), the heterologous bioorthogonal aminoacyl-tRNA synthase (aaRS)-tRNA pairs can add ncAAs to any site in a protein. 267 Many different aaRS/tRNA pairs have been developed. 268 , 269 , 270 The high-efficiency genetic code expansion devices allow the production of ncAA-containing protein and multiple ncAA-inserted proteins. 271 , 272 The ncAA insertions are succeed in all main model organisms. 273 , 274 Applications of the genetic code expansion system in medical fields are summarized here.

Genetic code expansion for antibody-drug conjugates

The antibody-drug conjugates (ADC) combine antigen-recognizing abilities of antibodies and tumor-killing capacities of chemicals commonly used in tumor therapies. 275 Traditional ADC drugs are chemical modification of cysteines or lysines in the antibodies, which may affect the immunogenicity, stability and half-life. 276 With the development of genetic code expansion technology, the introduction of a functional ncAA in the antibodies are feasible. 277 The site-specific, high-efficiency conjugation between antibodies and chemicals can be achieved. Oller-Salvia et al. developed a novel genetic code expansion system incorporating a cyclopropene derivative of lysine into antibodies. 278 The antibody conjugates to monomethyl auristatin E (MMAE) via a rapid Diels-Alder reaction. 278 The resulting ADC was stable and effective in serum. Wang et al. conjugated the Lck inhibitor dasatinib to monoclonal antibody CXCR4 using genetic code expansion methods. 279 The ADC avoids the side reactions during the chemical modification. The resulting dasatinib-antibody conjugate inhibited T-cell activation with low EC 50 with negligible effects on cell viability.

Genetic code expansion in the bispecific antibodies

Bispecific antibodies (BsAb) possess two specific antigen binding sites with enhanced tumor-killing abilities. 280 Some BsAbs have been approved by FDA. 281 The traditional BsAb production method relies on fusions of proteins, resulting in steric hindrance in the ligand-binding domains. 282 Additionally, the antibody production is at a low level with short half-life. 283 Synthesis of BsAbs via chemical modifications meets similar questions to ADC productions. 284 Genetic code expansion methods can conjugate two antibodies via a PEG linker to circumvent the challenges. Kim et al. introduced a ncAA (pAcF) to the antigen-binding fragment Fab region of anti-HER2 and anti-CD3 antibodies to form BsAb via two-step reactions. 285 Picomolar concentrations of the BsAb induced effector-cell mediated cytotoxicity in vitro. Employing the Diels-Alder reaction between tetrazine-containing ncAA and bicyclononyne- containing ncAA, a BsAb recognizing BCMA was developed to treat multiple myeloma, 286 successfully overcoming the drug-resistances in patients with multiple myeloma.

Genetic code expansion for engineering adeno-associated viruses (AAV)

AAVs are small parvovirus infecting human and primates. 287 AAVs are commonly used in gene therapies to achieve non-pathogenic, broad host range and high transfection and expression efficiencies. 288 However, the controllability and targeting ability are limited, hampering their applications. Zhang et al. used genetic code expansio to enhance the targeting ability of AAVs, conjugating cyclic arginyl-glycyl-aspartic acid (cRGD) to the shell protein of AAVs for targeting integrin. 289 Erickson et al. engineered AAVs for opto-control of the infection. 290 The R585 and R588 residues in vp1 protein of AAV2 were replaced by a light-sensitive ncAA, which hampered the interaction of vp1 and HSPG protein, resulting in inhibiting the infection of AAV. Exposed to UV light would remove the light-sensing moiety, recovered the infecting abilities of AAVs. 290 The method enhances time-spatial controllability of AAV vectors.

Genetic code expansion for prolonging a protein half-life

PEG is commonly used in prolonging the half-life of therapeutic proteins. 291 However, the random-modified PEG usually influences binding sites of therapeutic agents. 292 Thus, genetic code expansion may provide advantages in modifying proteins. Cho et al. used genetic code expansion to site-specifically modify PEG in human growth hormone, which is highly instable in clinical applications. 293 The modified human growth hormone is also with good batch to batch repeatability during the manufacturing processes. Some ncAAs increase protein stabilities per se. Xuan et al. demonstrated incorporation of a reactive isothiocyanate group into proteins to improve the heat-stability of myoglobin. Stable thiourea crosslinks were formed between the proteins. 294 Similar designs using long chain thiol-containing or fluorinated ncAAs were also verified. 295 , 296

Genetic code expansion for developing novel vaccines

ncAAs provide a wide variety of modifications of potential antigens that are candidates for vaccines. Gauba et al. inserted ncAAs containing nitrophenyl moiety into murine TNF-α protein for strong antibody response even with adjuvants. 297 ncAA-addicted genetically modified organism (GMO) is useful for vaccine developments. 298 The inactivated or attenuated pathogen-based vaccines usually have reduced effectiveness. 299 Construction of a GMO strain that relies on ncAA to survive has been conducted to amplify live-virus vaccines. By introducing a termination codon in the genome of influenza A virus, HIV-1 or hepatitis D virus, the viruses can only replicate in engineered cells with specific aaRS/tRNA pairs and ncAAs. Si et al. inserted a termination codon in the NP protein of influenza A viruses, leading to a stronger immunogenicity and triggering broader immune responses. 300 Based on the same idea, more and more live bacterial vaccines are under development. 298 However, bacteria are more complex compared to viruses. Many mutation mechanisms can help bacteria to escape from expression terminations. 301 The termination escapes restrict further applications with genetic code expansion in bacteria. Mandell et al. constructed a bacterium that metabolically dependent on ncAAs for survival. 302 The bacterium exhibited unprecedented resistance to evolutionary escapes, providing a hint to the development of live bacteria vaccines.

Other medical applications of genetic code expansion

The genetic code expansion technology can be applied for the construction of controllable CAR-T cells. Incorporation of p-azidophenylalanine (pAzF) into the Fab allows the identification and conjugation of fluorescein isothiocyanate (FITC), activating the antibody for cancer treatments. 303 Changing the inducer FITC to a short peptide was also proven applicable in cancer therapies. 304 FITC or peptides were used as inducers of CAR-T cells that provide a more safety-control approach for immunotherapies. The genetic code expansion has also been applied for biosynthesis of peptide natural products. Nisin is a complex lanthipeptide with broad-spectrum of anti-bacterial activities. Zambaldo et al. introduced a number of ncAAs into nisin, equipping it with novel macrocyclic topologies with enhanced activities. 305

The genetic code expansion methods are developing rapidly, modifying proteins both in vivo and site-specifically. The most sophisticated organism for this method is zebrafish and mouse. 306 The method should be improved to apply in more higher species. Although more than 200 different ncAAs have been used for genetic code expansion, most ncAAs are based on similar structural units. Enriching structure types is another direction for developments. In the future, genetic code expansion technology will bring more delicate treatments for mankinds.

Synthetic biology in the biosynthesis of therapeutic drugs

In the recent years, synthetic biology approaches has become promising in sustainable and cost-effective production of phamarceuticals. Synthetic biology designs (Fig. 5 ) and constructs biological circuits or chassis including bacteria, yeasts, cell cultures or whole plants, for effectively producing high-value added phamarceutical products or phamarceutical intermediates. It offers a scalable and sustainable way for productions of bioproducts using CO 2 based substrates, the production is rapid and robust, feasible for the large-scale industrial production, bioproducts can be manufactured without excessive cultivating and harvesting of medicinal plants (Table 1 ).

Technologies commonly used in synthetic biology. Various synthetic biology methods and tools have been developed to promote the design-build-test-learn cycle of cell factory construction, and these technologies are reforming the medical uses for synthetic biology. Pathway design is the first step, primary results are acquired via the constructed genetic circuits. Some optimizations are needed before next-round of tests, and the characteristics of the system is better understood from preliminary data. The design-build-test-learn cycles are iterative processes to improve robustness and efficacy of synthetic biology systems

As a classical field in synthetic biology, synthesis of pharmarceuticals is different from other medical applications. it generally uses yeast or bacteria as the production chassis. Synthetic biology concepts are extensively used in microorganisms, especially the DBTL (design-build-test-learn) (Fig. 5 ). DBTL cycle comprises the molecular biology designs and constructs in the beginning, and the experimental results are the basis for the new cycles of designs. The single-cell systems are easier to be manipulated than mammalian cells, In manmalian systems, the DBTL cycle can take very long, which is also an obstacle for mammalian synthetic biology. In the microbial synthesis of drugs, high-throughput screening and directed evolution are commonly used to accelerate experimental paces. Synthetic biology in microbes points to the direction of manmalian synthetic biology in a sense.

Biosynthesis of terpenoid drugs

Terpenoids are 5-carbon compound isoprene derivatives, also the largest group of plant secondary metabolites comprising approximately 60% of identified natural products. 307 Many of them are bioactive medical ingradients. 308 The anti-malaria drug, artemisinin, is sesquiterpene lactone containing an endoperoxide bridge. 309 Initially, artemisinin was extracted from the plant Artemisia annua 310 with a very low (0.01%-1%) content, 311 much less than the actual medical demands. The chemical route to artemisinin is difficult and inefficient mainly due to the multiple-chiral centers of this molecule. 312 The microbial synthesis of artemisinin prodrugs lowered drug cost. Biosynthesis of amorphadiene was a milestone in synthetic biology. The recombinant E. coli synthesized initially only 24 µg caryophyllene equivalent/ml. 9 After continuous optimizations, another artemisinin prodrug, namely, artemisinic acid, reached 25 g/L produced by engineered yeast. 22 , 23 The biosynthesis of artemisinic acid is a successful example of synthetic biology.

Taxol is a diterpene extracted from Pacific yew trees, serving as an anti-cancer agent. 313 Its production mainly relies on laborious and low-efficiency plant cell cultures. 314 Ajikumar et al. engineered E. coli cells to produce a taxol precursor, taxadiene, at a titer of 1 g/L. 315

The ginsenosides are triterpene saponins found in the plant genus Panax with cancer prevention and anti-aging effects. 316 Using the yeast cell-factory, various ginsenosides including ginsenoside Rh2 and ginsenoside compound K are synthesized with the titers of 2.2 g/L and 5.0 g/L, respectively. 317 , 318 Microbial approach reduces the shortage of ginsenoside for clinical uses.

Biosynthesis of alkaloid drugs

Alkaloids are a variety of organic compounds containing at least one nitrogen atom. 319 As a natural product, alkaloids are commonly used as they have pharmacological activities. 320 Biosynthesis of alkaloids circumvent the bans on growing certain plants like poppy and marijuana. 321 The formation of chiral centers during biosynthesis also outcompetes chemical synthesis for most chiral alkaloid compounds. 322 Galanie et al. employed engineered yeast cells to produce thebaine and hydrocodone. 323 Overexpression of 21 genes (for thebaine) or 23 genes (for hydrocodone) led to their formations of 6.6 × 10 −5 g/L and 3 × 10 −7 g/L, respectively. Nakagawa et al. improved the process using E. coli chassis. 324 The titers for thebaine and hydrocodone were enhanced to 2.1 × 10 −3 and 4 × 10 −5 g/L, respectively. The production of opiates reached miligram level. Subsequent metabolic engineering are needed to promote biosynthesized opiates to meet market demands.

Similar to the biosynthesis of artemisinic acid, cannabinoids are natural products from cannabis, commonly used for pain killing and anxiolytic actions. 325 (S)-Tetrahydropalmatine and cannabigerolic acid are two well-known cannabinoid hard to extract from plants. 326 The biosynthesis processes for cannabigerolic acid were established by Luo et al. The yield from yeast reached 0.1 g/L. 327 (S)-Tetrahydropalmatine biosynthesized by yeast by Hafner et al. reached 3.6 × 10 −6 g/L, a successful concept-of-proof for microbial production of complicated cannabinoids. 328

Biosynthesis of amino acid-derivative drugs

Using amino acids as building blocks, amino acid derivatives are also played an important role in human health. 329 This class of compounds is usually synthesized via biological routes rather than chemical synthesis for their multiple chirality moieties. Compared with alkaloid and terpenoids, amino acid-derivatives are more simple in structures with diversity. 329 Psilocybin is a L-tryptophan derivative with effects of anti-drug-addiction, relieving depression and anti-post-traumatic stress disorder effects. 330 E. coli or Saccharomyces cerevisiae have been engineered to heterologously express the synthetic pathways, forming 1.2 g/L and 0.6 g/L psilocybin, respectively. 330 , 331 Dencichine, also known as β- N -oxalyl- L -α,β-diaminopropionic acid (β-ODAP), is a plant metabolite first isolated from Lathyrus sativus seeds. Dencichine can induce platelet aggregation in human blood, and it is the main effective component of the Chinese medicine Yunnan Baiyao. 332 , 333 The authors optimized metabolic flux to dencichine in E. coli to the production with final titer reaching 1.29 g L −1 and a yield of 0.28 g g −1 glycerol. 334 Microbial production of dencichine exhibits an example of employing artificial enzymes and pathways to produce a desired chemical in synthetic biology applications.

Biocatalytic of asymmetric synthesis

Synthetic biology can assist multiple chiral-center chemical developments. Sitagliptin (Januvia) is a commonly used diabetes treatment, inhibiting DPP-4 enzyme in a competitive manner, reducing the cleavage of GLP-1 to increase the secretion of insulin. 335 The market of Januvia reached 1.4 billion dollars by 2021. 336 For chemical synthesis of sitagliptin, the chiral amine is transferred via a rhodium-based chiral catalyst with a low stereoselectivity and the product contaminated with rhodium. 337 A transaminase and synthetic-biology-based engineering approach based on homologous modeling and saturation mutagenesis, a process was developed that substantially improved the efficiency and purity for sitagliptin synthesis. 337

Cell-free synthetic biology in medical applications

Till now, efforts in synthetic biology have mainly focused on reprogramming organisms, development of genetic circuits and biological modules. However, because our knowledge on how life works is limited, the complex feature of creatures hindered progresses in synthetic biology. User-defined systems can solve the problem. Cell-free system is prepared to perform in vitro biological activities free from living cells ( e.g. tr anscription and translation). 338 As it is open, easy to control, flexible and high tolerance to cytotoxicity, 339 , 340 the system has been used in synthesizing proteins that are difficult to express or toxic in cells (Fig. 6 ). 341 Moreover, cell-free systems fit well to high-throughput screening. 342 Recently, with the development of cell-free biosensing diagnosis 343 and the advances in lyophilization, 344 the applications of cell-free synthetic biology have expanded into medical and pharmaceutical fields. 345

The charasteristics of cell-free synthetic biology. The types, advantages, products, and bottlenecks of cell-free systems are summarized in this figure. Generally, cell-free systems are used to produce pharmaceuticals or served as in vitro sensors. The main advantages are convenient, flexible and high tolerance to cytotoxicity. After solving the problems like high cost and instabilities, the system is promising for actual medical applications

Cell-free synthetic biology in pharmaceutical protein synthesis

Protein and peptide drugs are target-specific mostly with high activities and low toxicity for medical uses. 346 , 347 , 348 Many well-known drugs are proteins or peptides like Trastuzumab (Herceptin), 349 Adalimumab (Humira), 350 Insulin Glargine (Lantus) 351 and 13-valent pneumococcal conjugate vaccine (PCV13). 352 70% of the protein drugs are produced using the CHO cells. 353 However, some proteins are toxic for growth of cell hosts. 354 Cell-free protein synthesis (CFPS) provides a solution to the toxicity problems. 355 Additionally, screening of intracellular proteins are feasible in CFPS systems, 356 also lyophilization technologies allow the cell-free system to maintain highly active after one-year preservation. 357

The cell-lysate based- and purified component systems are two commonly used CFPS systems. 358 Theoretically, any organism could be used as the source in cell-lysate based system. The most common cell extract is from E. coli , wheat germ and yeast. 359 E. coli lysate is frequently used for protein synthesis, 360 wheat germ lysates for construction of protein arrays, 361 , 362 yeast lysates for synthesis of glycoproteins. 363 The purified component system comprises all purified translational-elements. Shimizu et al. developed a cell-free system using 36 transcription/translation related enzymes with highly purified ribosomes. 364 The system is efficient although minimum. However, the high cost of purified components hampers its applications. The cell-lysate based system is the first choice of CFPS systems.

Vaccination is the most effective way for pandemic prevention. 365 Cell-free systems provide a platform for rapid production of vaccines. Kanter et al. developed a cell-free system for highly effective production of a fusion protein consisting of a single chain Fv antibody fragment (scFv) connected to granulocyte-macrophage colony-stimulating factor (GM-CSF), a vaccine of B-cell lymphoma. 366 Lu et al. described a CFPS overexpressing a domain of pandemic H1N1 influenza virus for potentially and broadly protective influenza vaccines. 367 Besides bacterial systems, eukaryotic cell-free systems can express complex vaccines. Tsuboi et al. successfully expressed three malarial proteins in yeast lysate based cell-free systems, which is hard to produce in recombinant cells. 368

Antibodies are important for disease treatments and diagnosis. 369 CFPS is commonly used during the synthesis of antibodies. Ryabova et al. successfully produced functional scFv fragments in E. coli lysate-based cell-free system. 370 Post-translational modification (PTM) is the final maturation step of proteins. 371 Glycosylation is the main form of PTM important for maintaining the half-life and activity of protein drugs including some antibodies. 372 , 373 CFPS can also introduce functional PTM to proteins. Jaroentomeechai et al. used CFPS to synthesize N-glycosylated scFv using E. coli cell-free systems. 374 Overall, cell-free systems are useful complements to recombinant expressing systems for their rapid and on-demand properties.

Cell-free synthetic biology for diagnosis

Generally, detection of pathogens are based-on biosensors. 375 The sensing elements include enzymes, transcriptional factors, antibodies, organelles, whole-cells and tissues. 376 , 377 , 378 , 379 , 380 Although many biosensors are rapid and sensitive, the disadvantages are including the instability of enzymes, biosafety concerns of whole-cell biosensors and the complexity in preparing microfluidic sensors. 293 , 381 Therefore, cell-free sensors are developed. Pellinen et al. used luciferase as the reporter, Tet repressor and MerR regulatory proteins as the sensing elements, for the detection of tetracycline and the toxic mercury in cell-free systems. 382 Davies et al. constructed a cell-free protein array to screen high-immunogenicity proteins in human serums after virus infections, for the prophylactic uses and diagnosis. 383 In remote regions or harsh environments, cell-free systems lyophilized and attached on papers (or other matrices) are convenient and stable. 384 Pardee et al. employed lyophilized cell-free sensors to rapid determination of Ebola and Zika virus. 385 , 386 Future cell-free synthetic biology may lead to sophisticated design and synthesis of more complicated therapeutic agents, or rapid and sensitive biosensors for chronic disease diagnostics.

Discussion and future perspectives

Since the rapid developments started from more than a decade ago, synthetic biology has grown substantially and has emerged with many achievements, both in science and application aspects (Fig. 1 ). In this review, we summarized the advanced strategies and designs in synthetic biology for traditional pharmaceutical and medical applications, such as engineered smart cells (Fig. 2 ), 387 live probiotic therapeutics, 151 diagnostics, 388 stem cells, 83 drug production, 23 nanocarriers 389 and artificial vaccine developments. 300 The novel approach will enrich clinical regimens, shorten drug development cycle and lower pharmaceutical prices.

Synthetic biology approaches that most probably bring (or has brought) dramatic changes in biomedical fields include: the use of light for time-spartial controllable precise cell therapeutics (optigenetics), designed bacteria to target cancer cells, engineered cells rewiring metabolic flux in human or engineer the gut-brain-liver axis (engineered live therapeutics). Recent studies have shown possibilities that biosystems mentioned above are functioning well in manmalian and exhibiting considerable therapeutic effects in animal models or even volunteers. 70 However, they are just developed in their early stages. Many efforts are still needed to translate the lab findings to commercial products for patients.

The personalized engineered medicine is the next-gneration treatment strategy in the future. Smart therapeutics based on genetic-encoded circuits that can intepret environmental signal into effector activities will be commonly used. The auto-regulated therapeutic cells that sense diagnostic inputs for therapeutic outputs are one-station solutions for diagnosis, disease prevention and treatments (Fig. 2 ). Some applictions like CAR-T therapies have entered clinical stages, but most of the smart cells are not. Many attempts have failed in the early clinical, mainly for the low therapeutical abilities and unexpected side effects in human. Future works should emphasis on their safety as well as the efficacy and stability in treatments.

The combination of synthetic biology and artificial intelligence (AI) is promising to accelerate the advances both in medical and pharmaceutical fields, although the field is in initial stage. AI is a hit not only in computer science, but also in biology research. 390 The AI prediction of protein structures ranks as the top one in ten scientific breakthroughs in 2021. 391 The era of AI and big data is arriving, in-depth learning technique is advantageous in the characterization of complex objects, 392 fusion of multimodal features 393 and auto-sample generations. 394 AI can be applied in the synthetic biology field. At present, the combined applications of AI and synthetic biology have mainly been focused on the following three aspects, including, firstly, foresight of future research directions; collection of related synthetic biology data, then distinguish the casual link to analyze and evaluate the application and development directions. This is very helpful in analysis of numerous clinical datasets. Secondly, in the pharmaceutical applications, screening effective drugs based on AI and bioinformatic big data, testing candidate chemicals and simulating the therapeutic processes in disease models. It is a high-throughput method saving much manpower. Thirdly, development of novel drugs via reconstruction or modification the genomes by in-depth AI learning models, synthesizing novel compounds for drug discoveries. In the future, AI is promising to assist medical synthetic biology in designing more complicated systems (engineered cells or tissues) based on actual demands, substantially decreasing labor amounts of researchers.

However, some shortages and bottlenecks are to tackle for medical synthetic biology. Much effort is needed before the synthetic biology-based therapy become an available clinical option (Fig. 7 ). Although engineered cells containing genetic circuits are one of the most exciting designs in recent decades, they have limitations in actual uses of extracellular, signal-transduction free diseases which can be treated via traditional ways. 395 Tissue-specific engineered therapeutics are not succeed till now. The interferences of manmalian metabolisms are remain unknown. Solving these problems will be helpful for synthetic biology-based clinical applications.

The present situations, technical bottlenecks and future developments of synthetic biology based gene therapies. Some diagnosis and therapeutical approaches are available via rewiring metabolic and (or) signaling pathways in present synthetic biology. However, some bottlenecks like safety, versatility and efficacy are needing to tackle. Besides, novel designs such as AI-aided synthetic biology and rationally constructed live organisms and proteins are progressing

The majority of synthetic biology is still applied in microbes. However, most of the major issues, especially in solving human health problems, are needed for mammalian systems. Therefore, much efforts must be made for advancing mammalian synthetic biology to the next-generation therapeutic treatments, including the engineering of synthetic gene networks for disease treatments, tissue engineering or stem-cell generation and differentiation.

Additionally, synthetic biology-based therapeutics are still facing same social problems in ethical and legal fields similar to transgenic foods and stem cell therapies, although they can be imposed of better control from stringent pathways.

Even so, the future for synthetic biology-based therapeutics are promising, with new tools and applications developed in biomedical fields and highly-efficient microbial pharmaceutical production in the twenty-first century.

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Acknowledgements

This study was supported by grants from the Ministry of Science and Technology of China [Grant number 2018YFA0900200], National Natural Science Foundation of China [Grant number 32130001], Center of Life Sciences of Tsinghua-Peking University, the Shuimu Tsinghua Scholar Program and Chunfeng Foundation. This project is also funded by the National Natural Science Foundation of China [Grant numbers 31961133017, 31961133018]. These grants are part of MIX-UP, a joint NSFC and EU H2020 collaboration. In Europe, MIX-UP has received funding from the European Union’s Horizon 2020 research and innovation program [grant agreement Number 870294].

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Yan, X., Liu, X., Zhao, C. et al. Applications of synthetic biology in medical and pharmaceutical fields. Sig Transduct Target Ther 8 , 199 (2023). https://doi.org/10.1038/s41392-023-01440-5

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Published on 10.9.2024 in Vol 26 (2024)

Prompt Engineering Paradigms for Medical Applications: Scoping Review

Authors of this article:

• Jamil Zaghir 1, 2 * , MSc   ; 
• Marco Naguib 3 * , MSc   ; 
• Mina Bjelogrlic 1, 2 , PhD   ; 
• Aurélie Névéol 3 , PhD   ; 
• Xavier Tannier 4 , PhD   ; 
• Christian Lovis 1, 2 , MPH, MD  

1 Division of Medical Information Sciences, Geneva University Hospitals, Geneva, Switzerland

2 Department of Radiology and Medical Informatics, University of Geneva, Geneva, Switzerland

3 Université Paris-Saclay, CNRS, Laboratoire Interdisciplinaire des Sciences du Numérique, Orsay, France

4 Sorbonne Université, INSERM, Université Sorbonne Paris-Nord, Laboratoire d'Informatique Médicale et d'Ingénierie des Connaissances en eSanté, LIMICS, Paris, France

*these authors contributed equally

Corresponding Author:

Jamil Zaghir, MSc

Department of Radiology and Medical Informatics

University of Geneva

Chemin des Mines, 9

Geneva, 1202

Switzerland

Phone: 41 022 379 08 18

Email: [email protected]

Background: Prompt engineering, focusing on crafting effective prompts to large language models (LLMs), has garnered attention for its capabilities at harnessing the potential of LLMs. This is even more crucial in the medical domain due to its specialized terminology and language technicity. Clinical natural language processing applications must navigate complex language and ensure privacy compliance. Prompt engineering offers a novel approach by designing tailored prompts to guide models in exploiting clinically relevant information from complex medical texts. Despite its promise, the efficacy of prompt engineering in the medical domain remains to be fully explored.

Objective: The aim of the study is to review research efforts and technical approaches in prompt engineering for medical applications as well as provide an overview of opportunities and challenges for clinical practice.

Methods: Databases indexing the fields of medicine, computer science, and medical informatics were queried in order to identify relevant published papers. Since prompt engineering is an emerging field, preprint databases were also considered. Multiple data were extracted, such as the prompt paradigm, the involved LLMs, the languages of the study, the domain of the topic, the baselines, and several learning, design, and architecture strategies specific to prompt engineering. We include studies that apply prompt engineering–based methods to the medical domain, published between 2022 and 2024, and covering multiple prompt paradigms such as prompt learning (PL), prompt tuning (PT), and prompt design (PD).

Results: We included 114 recent prompt engineering studies. Among the 3 prompt paradigms, we have observed that PD is the most prevalent (78 papers). In 12 papers, PD, PL, and PT terms were used interchangeably. While ChatGPT is the most commonly used LLM, we have identified 7 studies using this LLM on a sensitive clinical data set. Chain-of-thought, present in 17 studies, emerges as the most frequent PD technique. While PL and PT papers typically provide a baseline for evaluating prompt-based approaches, 61% (48/78) of the PD studies do not report any nonprompt-related baseline. Finally, we individually examine each of the key prompt engineering–specific information reported across papers and find that many studies neglect to explicitly mention them, posing a challenge for advancing prompt engineering research.

Conclusions: In addition to reporting on trends and the scientific landscape of prompt engineering, we provide reporting guidelines for future studies to help advance research in the medical field. We also disclose tables and figures summarizing medical prompt engineering papers available and hope that future contributions will leverage these existing works to better advance the field.

Introduction

In recent years, the development of large language models (LLMs) such as GPT-3 has disrupted the field of natural language processing (NLP). LLMs have demonstrated capabilities in processing and generating human-like text, with applications ranging from text generation and translation to question answering and summarization [ 1 ]. However, harnessing the full potential of LLMs requires careful consideration of how input prompts are formulated and optimized [ 2 ].

Input prompts denote a set of instructions provided to the LLM to execute a task. Prompt engineering, a term coined to describe the strategic design and optimization of prompts for LLMs, has emerged as a crucial aspect of leveraging these models. By crafting prompts that effectively convey tasks or queries, researchers and practitioners can guide LLMs to improve the accuracy and pertinence of responses. The literature defines prompt engineering in various ways: it can be regarded as a prompt structuring process that enhances the efficiency of an LLM to achieve a specific objective [ 3 ] or as the mechanism through which LLMs are programmed by prompts [ 4 ]. Prompt engineering encompasses a plethora of techniques, often separated into distinct categories such as output customization and prompt improvement [ 4 ]. Existing prompt paradigms are presented in more detail in the Methods section.

In the realm of medical NLP, significant advancements have been made, such as the release of LLMs specialized in medical language and the availability of public medical data sets, including in languages other than English [ 5 ]. The unique intricacies of medical language, characterized by its terminological precision, context sensitivity, and domain-specific nuances, demand a dedicated focus and exploration of NLP in health care research. Despite these imperatives, to our knowledge, there is currently no systematic review analyzing prompt engineering applied to the medical domain.

The aim of this scoping review is to shed light on prompt engineering, as it is developed and used in the medical field, by systematically analyzing the literature in the field. Specifically, we examine the definitions, methodologies, techniques, and outcomes of prompt engineering across various NLP tasks. Methodological strengths, weaknesses, and limitations of the current wave of experimentation are discussed. Finally, we provide guidelines for comprehensive reporting of prompt engineering–related studies to improve clarity and facilitate further research in the field. We aspire to furnish insights that will inform both researchers and users about the pivotal role of prompt engineering in optimizing the efficacy of LLMs. By gaining a thorough understanding of the current landscape of prompt engineering research, we can pinpoint areas warranting further investigation and development, thereby propelling the field of medical NLP forward.

Study Design

Our scoping review was conducted following the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews) guidelines for scoping reviews (available in Multimedia Appendix 1 ). In this review, we use terminology to denote emerging technical concepts that lack consensus definitions. We propose the following definitions based on previous use in the literature:

• LLM: Object that models language and can be used to generate text by receiving large-scale language modeling pretraining (Luccioni and Rogers [ 6 ] define an arbitrary threshold at 1 billion tokens of training data). An LLM can be adapted to downstream tasks through transfer learning approaches such as fine-tuning or prompt-based techniques. Following the study of Thirunavukarasu et al [ 7 ] of models for the medical field, we include Bidirectional Encoder Representations From Transformers (BERT)–based and GPT-based models in this definition, although Zhao et al [ 8 ] place BERT models in a separate category.
• Fine-tuning: Approach in which the weights of the pretrained LLM are retrained on new samples. The additional data can be labeled and designed to adapt the LLM to a new downstream task.
• Prompt design (PD) [ 1 , 2 ]: Manually building a prompt (named manual prompt or hard prompt), tailored to guide the LLM toward resolving the task by simply predicting the most probable continuity of the prompt. The prompt is usually a set of task-specific instructions, occasionally featuring a few demonstrations of the task.
• Prompt learning (PL) [ 3 ]: Manually building a prompt and passing it to an LLM, trained via the masked language modeling (MLM) objective, to predict masked tokens. The prompt often features masked tokens, over which the LLM makes predictions. Those are then projected as predictions for a new downstream task. This approach is also referred to as prompt-based learning.
• Prompt tuning (PT) [ 9 ]: Refers to the LLM prompting where part or all the prompt is a trainable vectorial representation (known as continuous prompt or soft prompt) that is optimized with respect to the annotated instances.

Figure 1 illustrates the 4 approaches described above.

Inclusion and Exclusion Criteria

Studies were included if they met the following criteria: focus on prompt engineering, involvement of at least 1 LLM, relevance to the medical field (biomedical or clinical), pertaining to text-based generation (excluding vision-related prompts), and not focusing on prompting for academic writing purposes. Furthermore, as most of the first studies about prompt engineering emerged in 2022 [ 2 ], we added the following constraint: the publication date should be later than 2021.

Screening Process

The initial set of papers retrieved from the searches underwent screening based on titles, abstracts, and keywords. The search strategy is described in Multimedia Appendix 2 . Screening was performed by 2 reviewers (JZ and MN), working in a double-blind process. Interannotator agreement was calculated, with conflicts resolved through discussion.

Data Synthesis

We extracted information on prompt paradigms (PD, PL, and PT), involved LLMs, data sets used, studied language, domain (biomedical or clinical), medical subfield (if any), mentioned prompt engineering techniques, computational complexity, baselines, relative performances, and key findings. Additionally, we extracted journal information and noted instances of PD or PL or PT terminology misuse. Details are available in Multimedia Appendix 3 . Finally, we compile a list of recommendations based on the positive or negative trends we identify from the selected papers.

Screening Results

The systematic search across sources yielded 398 papers. Following the removal of duplicates, 251 papers underwent screening based on title, abstract, and keywords, leading to the exclusion of 94 studies. During this first screening step, 33 conflicts were identified and resolved among the annotators, resulting in an interannotator agreement of 86.8% (n=218). Subsequently, 157 studies remained, and full-text copies were retrieved and thoroughly screened. This process culminated in the inclusion of a total of 114 papers in this scoping review. The detailed process of study selection is shown in Figure 2 . Among the selected papers, 13 are from clinical venues, 33 are from medical informatics sources, 31 are from computer science publications, and 4 are from other sources. Notably, 33 of them are preprints.

Prompt Paradigms and Medical Subfields

Table 1 depicts the number of papers identified within each prompt paradigm along with their associated medical subfields. Some papers may simultaneously involve several (up to 2 in this review) prompt paradigms. Notably, PD emerged as the predominant category, with a total of 78 papers. These papers spanned across various medical fields, with a greater emphasis on clinical (including specialties) rather than biomedical disciplines. The screening yields 29 PL papers and 19 PT papers, with both paradigms maintaining a balanced distribution between biomedical and clinical domains. However, it is noteworthy that unlike PL and PT, PD encompassed a much broader spectrum of clinical specialties, with a particular interest in psychiatry.

Terminology Use

In our review, the consistency of terminology use around prompt engineering was investigated, particularly concerning its 3 paradigms: PD, PL, and PT. Across the papers, we meticulously tracked instances where the terminology was applied differently to the definitions used in the literature and described in the introduction. Notably, PL was used to refer to PD 4 times [ 12 , 13 , 67 , 86 ] and PT once [ 119 ], while PT was used 5 times to describe PL [ 88 , 96 , 97 , 99 , 114 ] and twice for PD [ 23 , 43 ]. Terminology inconsistencies were identified in only 12 studies. Consequently, while there remains some degree of inconsistency, a significant majority of 102 papers adhered to the definitions identified as commonly used terminology.

Language of Study

Considering the latest developments in NLP research encompassing languages beyond English [ 124 ], reporting the language of study is crucial. Several papers do not explicitly state the language of study. In some cases, the language can be inferred from prompt illustrations or examples. In the least informative cases, only the data set of the study is disclosed, indirectly hinting at the language.

Table 2 illustrates the language distribution among the selected papers, noting whether languages are explicitly mentioned, implicitly inferred from prompt illustrations, or simply not stated but implied from the used data set. The language used in 2 papers [ 60 , 68 ] remains unknown.

a Stated in the paper.

b Inferred from prompt figures and examples.

c Inferred from the data set.

Notably, English dominates with 84.2% (n=96) of the selected papers, followed by Chinese at 15.7% (n=18). Then, the other languages are relatively rare, often appearing in studies featuring multiple languages. It is worth mentioning that languages besides English are usually explicitly stated, with the exception of a paper studying Korean [ 63 ]. In total, the language had to be inferred from prompt figures and examples in 48 papers, all in English.

Choice of LLMs

Given the diverse array of LLMs available, spanning general or medical, open-source or proprietary, and monolingual or multilingual models, alongside various architectural configurations (encoder, decoder, or both), our study investigates LLM selection across prompt paradigms.

Figure 3 outlines prevalent LLMs categorized by prompt paradigms, though it is not exhaustive and only includes commonly encountered architectures. For example, while encoder-decoder models are absent in PT in Figure 3 , there are a few instances where they are used [ 95 , 110 ].

ChatGPT’s popularity in PD is unsurprising, given its accessibility. Models from Google, PaLM, and Bard (subsequently rebranded Gemini), all falling under closed models, are also prominent. Among open-source instruct-based LLMs, fewer are used, notably those based on LLaMA-2 with 7 occurrences.

In PL, encoder models, those following the BERT architecture, dominate, covering both general and specialized variants. There are occasional uses of decoder models like GPT-2 in PL-based tasks [ 103 , 105 ]. PT involves all model types, with a preference toward encoders. Further details on the models used are available in Multimedia Appendix 3 .

Topic Domain and NLP Task Trends

Figure 4 [ 16 , 20 , 26 , 41 , 47 , 88 - 123 ] illustrates the target tasks used in the PL and PT papers. PL-focused papers predominantly address classification-based tasks such as text classification, named entity recognition, and relation extraction, with text classification being particularly prominent. This aligns with the nature of PL, which centers around an MLM objective. Among other tasks, a study based on text generation [ 111 ] makes use of PL to predict masked tokens from partial patient records, aiming to generate synthetic electronic health records. Conversely, PT papers tend to exhibit a slightly broader range of tasks.

Figure 5 [ 10 - 87 ] presents the same analysis for PD-based papers. Unlike PL and PT, a prominent trend observed is that several studies focus on real-world board examinations. Notably, these studies predominantly center around tasks involving answering multiple-choice questions (MCQs). It is worth noting that although MCQs might be cast as a classification task, in practice, it is cast as a generation task using causal LLMs. It is interesting to note that none of the selected PD papers propose the task of entity linking, despite the clear opportunity of leveraging LLMs’ in-context learning ability for medical entity linking.

Prompt Engineering Techniques

We extensively investigated the used prompt techniques: among PD papers, 49 studies used zero-shot prompting, 23 used few-shot prompting, and 10 used one-shot prompting. Few shot tends to outperform in MCQs, but its advantage over zero shot is inconsistent in other NLP tasks. We propose a comprehensive summary of the existing techniques in Table 3 .

As shown in Table 3 , chain-of-thought (CoT) prompting [ 2 ] stands as the most common technique, followed by the persona pattern. In medical MCQs, various attempts with CoT can lead to different reasoning pathways and answers. Hence, to improve accuracy, 2 studies [ 19 , 20 ] used self-consistency, a method involving using multiple CoT prompts and selecting the most frequently occurring answer through voting.

Flipped interaction was used for simulation tasks, such as doctor-patient engagement [ 60 ] or to provide clinical training to medical students [ 81 ]. Emotion enhancement was applied in mental health contexts [ 58 , 60 ], allowing the LLM to produce emotional statements.

More innovative prompt engineering techniques include k-nearest neighbor few-shot prompting [ 19 ] and pseudoclassification prompting [ 78 ]. The former uses the k-nearest neighbor algorithm to select the k-closest examples in a large annotated data set based on the input before using them in the prompt, and the latter presents to the LLMs all possible labels, asking the model to respond with a binary output for each provided label. Despite its potential, tree-of-thoughts pattern use was limited, with only 1 instance found among the papers [ 77 ].

Emerging Trends

Figure 6 illustrates a chronological polar pie chart of selected papers and their citation connections, identifying five highly cited papers: (1) Agrawal et al [ 40 ] demonstrate GPT-3’s clinical task performance, especially in named entity recognition and relation extraction through thorough PD. (2) Kung et al [ 36 ] evaluate ChatGPT’s (GPT-3.5) ability for the United States Medical Licensing Examination, shortly after the public release of ChatGPT. (3) Singhal et al [ 20 ] introduce MultiMedQA and HealthSearchQA benchmarks. The paper also presents instruction PT for domain alignment, a novel paradigm that entails learning a soft prompt prior to the LLM general instruction, which is usually written as a hard prompt. Using this approach on FlanPaLM led to the development of Med-PaLM, improving question answering over FlanPaLM. (4) Nori et al [ 27 ] evaluate GPT-4 on the United States Medical Licensing Examination and MultiMedQA, surpassing previous state-of-the-art results, including GPT-3.5 and Med-PaLM. (5) Luo et al [ 26 ] release BioGPT, a fine-tuned variant of GPT-2 for biomedical tasks, achieving state-of-the-art results on 6 biomedical NLP tasks with suffix-based PT.

Trends in PD

As shown in Figure 6 , the PD paradigm presents multiple trends: all papers disseminated in clinical-based venues, and 27 of 33 (82%) of the encountered preprints adhere to this paradigm. Furthermore, we observed a significant focus on work involving frozen LLMs within the PD domain. This trend is likely due to the frequent use of ChatGPT in 74 instances, as depicted in Figure 3 , despite OpenAI offering fine-tuning capabilities for the model. It is worth mentioning that 46 of 78 (59%) PD papers do not include any baseline, including human comparison. This gap will be further explored in a subsequent section.

Trends in PL and PT

Among PL and PT papers, computer science and medical informatics are the most prevalent venues. Although PL has drawn attention to the idea of adapting the MLM objective to downstream tasks without needing to further update the LLM weights, many studies still opt to fine-tune their LLMs, with a nonnegligible amount of them evaluating in few-shot settings [ 89 , 92 , 93 , 112 ]. Unlike PD, PL and PT usually include a baseline, with it often being a traditional fine-tuning version of the evaluated model [ 92 , 93 , 95 ] to compare it against novel prompt-based paradigms. These studies came to a common conclusion, being that PL is a promising alternative to traditional fine-tuning in few-shot scenarios.

There are 2 ways for conducting PL: one involves filling in the blanks within a text, known as cloze prompts, while the other consists in predicting masked tokens at the end of the sequence, referred to as prefix prompts. A distinct advantage of the latter approach is its compatibility with autoregressive models, as they exclusively predict the appended masks. Among the 29 PL papers, 21 (72%) of them propose cloze prompts, while 15 (52%) use prefix prompting. The involved NLP tasks are well-distributed across these 2 prompt patterns. Another crucial component of PL is the verbalizer. As PL revolves around predicting masked tokens, classification-based tasks require mapping manually selected relevant tokens to each class (manual verbalizer). Alternatively, some studies propose a soft verbalizer, akin to soft prompts, which automatically determines the most relevant token embedding for each label through training. Of the 29 PL papers selected, 16 (55%) studies explicitly mention the use of a manual verbalizer, while 2 explored both verbalizers to assess performance [ 101 , 110 ]. Only 1 exclusively used a soft verbalizer [ 89 ]. Another study does not use any verbalizer, as it focuses on generating synthetic data by filling the blanks [ 111 ]. Notably, 8 (28%) studies did not report any mention regarding the verbalizer methodology.

Hard prompts, which are related to PD and PL, involve manually crafted prompts. Regarding PT, optimal prompts are attainable through soft prompting (ie, prompts that are trained on a training data set), yet, determining the appropriate soft prompt length remains obscure. In total, 5 of 19 (26%) PT studies tried various soft prompt lengths and reported their corresponding performances [ 26 , 105 , 118 , 119 , 122 ]. While there is no definitive optimal prompt length, a trend emerges: optimal soft prompt length typically exceeds 10 tokens. Surprisingly, 8 (42%) papers omit reporting the soft prompt length. Regarding the placement of soft prompts in relation to the input and the mask, consensus is lacking. A total of 5 (26%) papers prepend the soft prompt at the input’s outset, while 4 (21%) append it as a suffix. One paper uses both strategies in a single prompt template [ 95 ]. Some innovative methods involve inserting a single soft prompt for each entity that needs to be identified in entity-linking tasks or using token-wise soft prompts, where each token in the textual input is accompanied by a distinct soft prompt. The position of soft prompts remains unreported in 5 (26%) studies. Finally, according to the 6 (32%) studies that used mixed prompts [ 90 , 91 , 95 , 101 , 105 , 110 ] (a combination of hard and soft prompts), it has consistently been reported that mixed prompts lead to a better performance than hard prompts alone.

Baseline Comparison

Only 62 of the screened papers reported comparisons to established baselines. These include traditional deep learning approaches (eg, fine-tuning approach), classical machine learning algorithms (eg, logistic regression), naive systems (eg, majority class), or human annotation. The remaining papers solely explored prompt-related solutions, without including baseline comparisons. Tables 4 - 6 traces the presence of a nonprompt baseline among different prompt categories ( Table 4 ), papers sources ( Table 5 ), and NLP tasks addressed ( Table 6 ).

a Higher or lower indicates that the performance of the proposed prompt-based approach is higher or lower than the baseline.

a NLP: natural language processing.

b Higher or lower indicates that the performance of the proposed prompt-based approach is higher or lower than the baseline.

Nonprompt-related baselines are often featured in studies focused on PL and PT but not PD. Additionally, PL and PT have a tendency to perform better than their respective reported baselines, PD tends to report less conclusive results. More specifically, among the 22 papers using either PL or PT with an identical fine-tuned model as a baseline, 17 indicate superior performance with the prompt-based approach, 3 observed comparable performance, and 2 studies noted inferior performance.

Significantly, papers from computer science venues tend to include more state-of-the-art baselines than those from medical informatics and clinical venues. Specifically, all 13 papers reviewed from clinical venues did not use any nonprompt baselines. Furthermore, there appears to be no consistent link between the type of NLP tasks and the omission of baselines, indicating that the decision to include baselines is more influenced by the evaluation methodology than by feasibility.

Prompt Optimization

Numerous studies in the literature highlight the few-shot learning capabilities of LLMs, often referred to as “few-shot prompting,” wherein they demonstrate proficiency in executing tasks with minimal demonstrations provided, typically through text prompts. However, it is crucial to acknowledge that the annotation cost associated with such frameworks might extend beyond the few annotated demonstrations within the prompt. Many studies claiming to explore few-shot or zero-shot learning through prompt engineering rely on extensive annotated validation data sets to refine PD and formulation. This is, for example, the case in the paper that popularized the term “few-shot learning” [ 1 ]. Among the 45 analyzed papers concentrating on few-shot or zero-shot learning, 5 explicitly detail the optimization of prompt formulation using extensive validation data sets. Conversely, 18 of these papers either do not engage in prompt optimization or test various prompts and document all results. Notably, 22 papers present results using only 1 prompt choice, without clarifying whether this choice was made thanks to additional validation data sets.

Summary of the Findings

This scoping review aimed to map the current landscape of medical prompt engineering, identifying key themes, gaps, and trends within the existing literature. The primary findings of this study reveal a greater prevalence of PD over PL and PT, with ChatGPT dominating the PD domain. Additionally, many studies omit nonprompt-based baselines, do not specify the language of study, or exhibit a lack of consensus in PL (prefix vs cloze prompt) and PT settings (soft prompt lengths and positions). English is notably dominant as the language of study. These findings suggest that while the field is emerging, there is a pressing need for improved research practices.

Costs, Infrastructure, and LLMs in Clinical Settings

Prompt engineering techniques enable competitive performance in scenarios with limited or no resources as well as in environments with low-cost computing infrastructure. As hospital data and infrastructure are often found in this scenario, these approaches hold great promise in the clinical field. Figure 6 shows the absence of PL- and PT-related works in clinical journals. This trend may stem from the widespread accessibility of ChatGPT, favoring PD-focused investigations. Despite efforts like OpenPrompt [ 125 ] to facilitate PL and PT works, the programming barrier likely deters clinical practitioners. Surprisingly, 7 papers use ChatGPT with sensitive clinical data. Despite the recent availability of ChatGPT Enterprise in GPT-4 for secure data handling, it is apparent that most of these studies have not used this feature since they used GPT-3.5. Limited use of local LLMs, especially LLaMA-based, suggests a need for their increased adoption in future clinical PD studies. The lack of local LLMs may be due to clinicians’ limited computational infrastructure.

Prompt Engineering Techniques Effectiveness in Medical Research

In documented prompt engineering techniques, the effectiveness of few-shot prompting compared to zero shot varies by task and scenario. However, CoT shows superior reasoning performance, compelling LLMs to present reasoning pathways and consistently outperforming zero-shot and few-shot methods across PD studies. Its ensemble-based variant, self-consistency, consistently outperforms CoT. Despite the persona pattern’s frequent use, there is a lack of ablation studies on its impact on medical task performance, with only 1 paper reporting negligible improvement [ 61 ]. Prompt engineering is an emerging field of study that still needs to prove its efficacy. However, almost half of the papers focused only on prompt engineering and failed to report any nonprompt-related baseline performance, despite the availability of such baselines for the addressed NLP tasks. On the whole, the results are far from being systematically in favor of LLM-based methods, greatly attenuating the impression of a technological breakthrough that is generally commented on. Selecting a baseline remains a necessary step toward understanding the actual impact of prompt engineering.

Bender Rule

Regarding the languages, while Table 2 shows the dominance of English in medical literature, many papers studying English fail to explicitly mention the language of study. This oversight is more prevalent in computer science and clinical venues, whereas medical informatics exhibits a more favorable trend, as validated by a chi-square test yielding a P value of .02 (Table S1 in Multimedia Appendix 2 ). Notably, languages such as Chinese are consistently mentioned across the 18 selected papers. However, the Bender rule, namely “always name the language(s) you are working on,” seems to be well respected for languages other than English. This finding has already been documented for NLP research in general [ 126 ].

Fine-Tuning Versus Prompt-Based Approaches

While traditional LLM fine-tuning remains a viable method for various NLP tasks, PL and PT are competitive alternatives to fine-tuning, particularly in resource-constrained and low computational scenarios. PL, leveraging predefined prompts to guide model behavior, offers an efficient approach in low-to-no resource environments. Conversely, PT emerges as a viable solution in low computational scenarios, as it requires substantially fewer trainable parameters compared to traditional fine-tuning approaches. Since both prompt-based approaches do not require the LLM to be further trained, they are less prone to catastrophic forgetting [ 127 ].

Recommendations for Future Medical Prompt–Based Studies

For future research in prompt engineering, we propose several recommendations aimed at improving research quality, reporting, and reproducibility. From this review, we identified several trends such as the computational advantages or the lack of evaluations on baselines with a lack of ablation studies to evaluate the performance of the prompting strategies. Some studies do not clearly mention the prompt engineering choices they made. For instance, in PL, choices range from using cloze to prefix prompting and from using manual to soft verbalizer. Similarly, PT is characterized by configurations of soft prompts, such as the length and the positions. To clarify these distinctions and enhance methodological transparency and reproducibility in future research, we have developed reporting guidelines available in Textbox 1 . Adhering to these reporting guidelines will contribute to advancing prompt engineering methodologies and their practical applications in the medical field.

General reporting recommendations

• For sensitive data, local large language models (LLMs) should be preferred to the ones that use an application programming interface or a web service.
• The language of the study used should be explicitly stated.
• The mention of whether the LLM undergoes fine-tuning should be made explicit.
• The prompt optimization process and results should be documented to ensure transparency, whether it is through different tested manual prompts or through a validation data set.
• The terms “few-shot,” “one-shot,” and “zero-shot” should not be used in settings where the prompts have been optimized on annotated examples.
• Experiments should include baseline comparisons or at least mention existing results, particularly when data sets originate from previous medical challenges or benchmarks.

Specific to prompt learning and prompt tuning

• Concepts (such as prompt learning and prompt tuning) should be defined and used consistently with the consensus.
• In prompt learning experiments, the verbalizer used (soft and hard) should be explicitly specified, or a clear justification should be provided if the verbalizer is omitted. Additionally, whether the prompt template follows the cloze or the prefix format should be mentioned.
• In prompt tuning experiments, authors should provide details on soft prompt positions, length, and any variations tested, such as incorporating hard or mixed prompts, as part of the ablation study.

Limitations

A limitation was the large number of papers retrieved during the initial search, which was addressed by limiting the search scope to titles, abstracts, and keywords. Furthermore, since some studies may perform prompt engineering techniques without mentioning any of the 4 prompt-related expressions used in the queries, they might be missed by our searches.

Conclusions

Medical prompt engineering is an emerging field with significant potential for enhancing clinical applications, particularly in resource-constrained environments. Despite the promising capabilities demonstrated, there is a pressing need for standardized research practices and comprehensive reporting to ensure methodological transparency and reproducibility. Consistent evaluation against nonprompt-based baselines, prompt optimization documentation, and prompt settings reporting will be crucial for advancing the field. We hope that a better adherence to the recommended guidelines, in Textbox 1 , will improve our understanding of prompt engineering and enhance the capabilities of LLMs in health care.

Acknowledgments

JZ is financed by the NCCR Evolving Language, a National Centre of Competence in Research, funded by the Swiss National Science Foundation (grant # 51NF40_180888).

Authors' Contributions

JZ and MN performed the screening and data extraction of the papers and synthesized the findings. AN and XT supervised MN. MB and CL supervised JZ. JZ and MN wrote the manuscript with support from MB, AN, XT, and CL. All authors contributed to the analysis of the results. CL conceived the original idea.

Conflicts of Interest

CL is the editor-in-chief of JMIR Medical Informatics . All other authors have no conflict of interest to declare.

PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews) checklist.

Search strategy and statistical analysis.

Reading notes and details of the reviewed papers.

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Abbreviations

Edited by T de Azevedo Cardoso; submitted 14.05.24; peer-reviewed by B Bhasuran, D Hu, A Jain; comments to author 03.07.24; revised version received 09.07.24; accepted 22.07.24; published 10.09.24.

©Jamil Zaghir, Marco Naguib, Mina Bjelogrlic, Aurélie Névéol, Xavier Tannier, Christian Lovis. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 10.09.2024.

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