Improvement of the Approval System for the Development and Experimentation of Living Modified Organisms in Synthetic Biology

Abstract

The advancement of synthetic biology and its expanding influence on future industries has given rise to a growing need to deregulate the development and experimentation of living modified organisms (LMOs) created by synthetic biology. This article aims to explore the current state of laws and regulations governing synthetic biology-related LMOs and identify trends in the development and experimentation of LMOs. Based on this analysis, we propose a plan for the improvement of the national approval system for LMO development and experimentation that both ensures the safety of LMO research and revitalizes related research.

Research Trends in Synthetic Biology

Recombinant DNA technology, which was developed in 1972, enabled the production of living modified organisms (LMOs). Since then, various molecular biology technologies, such as DNA sequencing, DNA synthesis, and gene editing, have been rapidly developed, resulting in increased sophistication, cost reduction, and acceleration of genetic recombination technologies. Consequently, the term synthetic biology emerged in the early 2000s. Synthetic biology involves the creation of new gene expression circuits that operate in cells (animals, plants, and microorganisms) or cell-free environments through componentization and redesign of the DNA parts required for gene expression [1]. This technology is used to produce various useful substances in living organisms including medical and industrial proteins and chemicals. It is also used in cell therapies, including microbial and immune cell therapies, as well as in the development of diagnostic systems. Its applications extend to various industries, including medicine, pharmacy, food, environment, and energy, and are expanding to other fields, such as agriculture.

Synthetic biology utilizes the DBTL (Design- Build-Test-Learn) cycle as its fundamental principle for design, and has introduced an automated system known as biofoundry [2]. This system has dramatically reduced both the time and cost of development, resulting in increased efficiency. Governments worldwide, such as those in the United States, the United Kingdom, and China, have identified synthetic biology as a core field of national competitiveness and are implementing aggressive policies and investments to promote and strategically develop synthetic biology. Furthermore, there has been significant investment in synthetic biology from the private sector, resulting in the emergence of numerous start-ups in this field. The synthetic biology market is predicted to grow rapidly as the global demand for sustainable and eco-friendly solutions increases. A report by the Boston Consulting Group in 2022 forecasts that synthetic biology will be widely adopted in manufacturing over the next decade, accounting for more than one-third of global production and will amass an estimated worth of USD 30 trillion [3]. As a result, discussions are ongoing regarding the need for new systems and laws to regulate the development and safe application of synthetic biology-related LMOs [4-6].

Trends in Laws and Regulations Related to Synthetic Biology-related LMOs

Currently, laws and regulations governing synthetic biology-related LMOs are based on the Cartagena Protocol on Biosafety, which was adopted in January 2000 as an annexed protocol for the Convention on Biological Diversity (CBD). This protocol is an international agreement designed to prevent potential harm from the transboundary movement, handling, and use of LMOs. To implement the Convention, Republic of Korea (ROK) enacted the LMO Act in March 2001 and became a party to the Protocol in October 2007. The Biosafety Protocol and LMO Act were enforced in ROK in January 2008. As of May 2023, 173 countries have become parties to the Protocol, while the United States, Russia, Australia, Canada, Argentina, and Singapore are among the major nonparties.

The Cartagena Protocol defines LMOs as any living organism that possesses a “novel combination of genetic material” obtained through the use of “modern biotechnology”. “Modern biotechnology” encompasses a range of techniques, including (1) in vitro nucleic acid techniques that include recombinant DNA methods and direct injection of nucleic acid into cells or organelles, or (2) fusion of cells beyond the taxonomic family, that overcome natural physiological, reproductive or recombination barriers and that are not techniques used in traditional breeding and selection [7].

Discussions are ongoing regarding whether products developed through the application of synthetic biology fall under the definition of “LMOs” as outlined in the Cartagena Protocol, and whether synthetic biology should be considered a form of “modern biotechnology.” Synthetic biology-related matters were first raised at COP9 (9th Conference of the Parties) of the CBD in 2008, and a special technical advisory group (Ad Hoc Technical Expert Group, AHTEG) on synthetic biology was established at COP10 in 2010 to collect experiences and information from each country through forums. Discussions on synthetic biology within the CBD have focused on various topics including gene drives, digital sequence information, risk assessment methods and procedures, Research and Development (R&D) monitoring, and social factors, such as biosecurity, bioethics, social acceptability, and information-sharing methods. One of the major topics of discussion within the CBD is whether synthetic biology meets the criteria for New and Emerging Issues (NEIs). As a result of these discussions, the CBD defines synthetic biology as “a further development and new dimension of modern biotechnology that combines science, technology and engineering to facilitate and accelerate the understanding, design, redesign, manufacture and/or modification of genetic materials, living organisms and biological systems, which is presented in a comprehensive sense corresponding to the overall modern life science technology.”

At COP13 in 2016, the AHTEG presented the conclusion that “living organisms developed through current applications of synthetic biology, or that are currently in the early stages of R&D are similar to LMOs as defined in the Cartagena Protocol.” This conclusion has been subject to ongoing debate, as some stakeholders argue that synthetic biology may require new or adapted regulatory frameworks because of its unique characteristics and potential risks. However, the main view is that LMOs made using synthetic biology can be regarded as subject to the current LMO regulations [7].

Domestic LMO Development: Experimentation Approval System and LMO Development Status

Since the enactment of the LMO Act, significant progress has been made in the development of synthetic biology, methods and targets of LMO research, and the frequency of LMO research. However, the current LMO development∙experimentation approval system is an obstacle to an otherwise thriving industry. As LMO development∙experimentation for both basic research and industrial applications is actively conducted, there is a growing demand for the improvement of LMO development∙experimentation approval laws to reflect the current level of technology and the opinions of researchers.

In ROK, according to the LMO Act, the development∙experimentation of LMOs are classified into four categories based on the degree of risk, exemption, institutional report, institutional approval, and national approval experiments (Figure 1) [8]. National approval experiments refer to experiments that require prior approval from the Korea Disease Control and Prevention Agency (KDCA) after institutional approval, and fall into four categories (Table 1).

Figure 1. Current biosafety review process.

Reused from Korea Disease Control and Prevention Agency (KDCA). Institutional Biosafety Committee (IBC) organization & operating guide. KDCA; 2021 [8].

Table 1. Targets for national approval for development·experimentations of LMOs.

Targets for national approval		Note
1	In the case of using a microorganism whose species name is not specified and whose pathogenicity to humans is unknown
2	In the case of using a gene having the ability to produce a protein toxin with a 50% lethal toxin amount of less than 100 ng per 1 kg of body weight for vertebrates	① Botulinum toxin (types A, B, C, D, E, F), ② Tetanus toxin ③ Heterogeneous neurotoxin, ④ Diphtheria toxin, ⑤ Toxins with 50% lethal toxin per 1 kg of body weight for other vertebrates is less than 100 ng
3	Intentionally transferring drug resistance genes to microorganisms in a way that does not occur naturally	Exemption if the following 8 types of resistance genes are used in an approved host-vector system judged to be highly safe Approved host-vector system ① Escherichia coli K12 or E. coli B; ② Bacillus subtilis or Bacillus licheniformis; ③ Saccharomyces cerevisiae; ④ Pseudomonas putida; ⑤ Streptomyces coelicolor, Streptomyces lividans, Streptomyces parvulus, Streptomyces griseus; ⑥ Neurospora crassa ⑦ Agrobacterium tumefaciens Exemped drug resistance genes ① Ampicillin, ② Chloramphenicol, ③ Hygromycin, ④ Kanamycin, ⑤ Streptomycin, ⑥ Tetracycline, ⑦ Puromycin, ⑧ Zeocin
4	In the case of direct use of the gene of a pathogenic microorganism requiring state management for national health reasons, or synthesis of the gene of the pathogenic microorganism concerned	Bacteria Vibrio cholerae O1·O139, Yersinia pestis, Bacillus anthracis etc. Eukaryotic microorgnisms Coccidioides immitis, Coccidioides posadasii Viruses and prions Herpes B virus, Ebola virus, Avian influenza infectious disease virus (H5N1, H7N7, H7N9) etc.

LMO=living modified organism.

According to data from the KDCA the number of approvals for LMO development∙experimentation subject to national approval increased from 35 in 2017 to 73 in 2021, with 281 cases approved over the past five years. Corporate research institutes accounted for the highest number of approvals (134 cases), followed by universities (78 cases), and national and public institutions (69 cases). It appears that R&D for commercialization follows a more thorough national approval process.

Among the categories reviewed, the introduction of drug resistance genes was the most frequent, with 196 cases among the four types of LMO development∙experimentation subject to national approval, followed by the use of pathogenic microorganisms in 74 cases and the use of pathogenic toxin genes in 37 cases. The drug resistance genes used were, neomycin in 124 cases, erythromycin in 26 cases, spectinomycin in 23 cases, gentamicin in 15 cases, and blasticidin and sulfadiazine in 8 cases each [9].

A survey was conducted in July 2022 with 76 synthetic biology researchers comprising 57 researchers from domestic universities, 10 from national and public research institutes, and 9 from corporate research institutes to strategize how to improve the national approval system for LMO development and experiments. The survey covered topics such as the actual state of LMO development research, safety management in LMO development research, national approval regulations for LMO development and experimentation, and opinions on how to improve these regulations (Suppl 1). The survey found that many researchers have used various prokaryotic and eukaryotic organisms for LMO production, in addition to the exempt host-vector system. The majority of LMO studies targeted low-risk microorganisms, with the risk group 1 (RG1) accounting for 63.2% and the risk group 2 (RG2) accounting for 44.7% of the strains used. According to the definition in the Recombinant DNA Guidelines, RG1 organisms do not cause disease in healthy adults, while RG2 organisms do not cause serious diseases and these diseases can be easily prevented or treated in humans. Eight new drug resistance genes including erythromycin, gentamicin, neomycin, and spectinomycin were used. LMO production showed a diverse distribution, with 75.0% of laboratories producing less than 1–10 cases per month, and 11.8% producing 10 or more cases per week.

Most researchers responded that strict measures were taken to prevent LMO leakage, but 34.2% did not accurately understand the national LMO approval regulations. The difficulties in applying for national approval were also highlighted, with research delays (68.2%) and difficulties in the approval application process (63.6%) accounting for a high percentage of responses. Finally, most researchers aim to relax some restrictions of the national approval system in order to advance development and experimentation in synthetic biology [9].

Approval Systems for LMO Development and Experimentation in Major Foreign Countries

Similar to ROK, major foreign countries categorize LMO development and experimentation into exemption, institutional reporting or approval, and state-approved testing; however, each country has different specific targets for synthetic biology application and they generally apply more relaxed regulations than ROK. Regulations for LMO development were examined in regions like the United States, the United Kingdom, and Singapore, where LMO development research is actively being conducted.

1. The United States

According to the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, established in 1994 and revised in 2019, various regulations govern the safety of basic and medical research involving animals, plants, microorganisms, and viruses. The Institutional Biosafety Committee (IBC) Registration Management System was integrated to manage the biosafety of genetic recombination experiments. The NIH guidelines classified these experiments into major and minor actions, and six types of experiments were defined. The regulations were divided into exemptions, IBC notification or approval, NIH Office of Science Policy (OSP) approval, and NIH Director approval, depending on the targets and categories of the experiments (Table 2) [10].

Table 2. Subjects for LMO development and experimentation approval according to the US NIH guidelines.

Major action			Minor action
A	B	C	D	E	F
NIH Director, IBC approval	NIH OSP, IBC approval	IBC approval	IBC approval	IBC report	Exemption
Cases in which drug resistance genes are intentionally introduced into microorganisms to make it difficult to treat diseases of humans, animals, and crops	Toxin genes with LD50 (50% lethal dose of toxin per kg of body weight for vertebrates) of less than 100 ng	HGT research	In case of using microorganisms or viruses higher than the RG2	Recombination or synthesis of less than 2/3 of the viral genome	Eight cases including Escherichia coli K-12, Saccharomyces, Kluyveromyces, Bacillus subtilis, Bacillus licheniformis

LMO=living modified organism; NIH=National Institutes of Health; IBC=Institutional Biosafety Committee; OSP=Office of Science Policy; HGT=human gene transfer; RG2=risk group 2.

Compared with Korean LMO development∙experimentation approval standards, the US NIH national approval is required for cases where there is a verifiable risk to humans, such as the introduction of a drug resistance gene in difficult-to-treat pathogenic microorganisms or when a highly toxic gene with an LD50 of less than 100 ng is used. In most cases, research can be conducted with the IBC’s institutional approval and reporting by implementing negative regulations, providing convenience and excellence in research. While positive regulations are being implemented for IBC agency approval and reporting exemptions, specifying approved host vector systems similar to domestic standards, the categories are broad, specific, and continuously added to the fields.

2. Singapore

The Singaporean government established the Genetic Modification Advisory Committee (GMAC) in April 1999 to oversee and provide guidance on genetically modified organisms (GMOs). In 2013, “Singapore Biosafety Guidelines for GMO Research Guidelines for Research on Genetically Modified Organisms” were introduced and subsequently revised in 2021. These guidelines have a structure similar to those of the US NIH guidelines and classify GMOs into three categories. IBC approval reviews are conducted only when necessary (Table 3), and the GMAC reviews and endorses the IBC’s approval documents.

Table 3. Subjects for approval of LMO development and experimentation according to the Singapore Biosafety Research Guidelines.

Category A (high risk)	Category B (low risk)	Category C (no risk)
IBC approval, GMAC endorsement	IBC approval	IBC report
Toxin genes with LD50 (50% lethal dose of toxin per kg of body weight for vertebrates) of less than 100 ng	Animals and plants	Model organism
	Unapproved host-vector system	Approved host-vector systems
Pathogenic viruses and microorganisms	Genes from pathogenic viruses and microorganisms

LMO=living modified organism; IBC=Institutional Biosafety Committee; GMAC=Genetic Modification Advisory Committee.

3. United Kingdom

The United Kingdom has amended The Genetically Modified Organisms (Contained Use) Regulations for confined use five times since 2000, and the 2014 amendment is currently being employed. This regulation, together with the EU Directive on the Restricted Use of Genetically Modified Microorganisms (2009/41/EC), sets out a comprehensive set of directives relating to the use of GM animals and plants. Contained use implies that the GMOs are developed in facilities that are physically, chemically, or biologically isolated from the environment. Relevant guidelines stipulate and present specific confined use conditions, that is, appropriate containment levels according to the level of risk classified through risk assessment. In addition, studies that required reporting or approval from the UK Health and Safety Executive were classified according to the risk level, as shown in Table 4. While most studies are classified as Class 1 in the guidelines, follow-up studies can begin without notification [11].

Table 4. UK Biosafety Guidelines.

Risk level	Risk level	Optimal level of containment	First report	Follow-up report
Class 1	No risk or negligible	Containment level 1	First user of the institution	Not needed
Class 2	Low risk	Containment level 2	Report	Report
Class 3	Medium risk	Containment level 3	Approval	Approval
Class 4	High risk	Containment level 4	Approval	Approval

The UK Safety and Health Agency appointed the Scientific Advisory Committee on Genetic Modification (SACGM), which provides detailed guidelines on GMO safety in the SACGM Compendium of Guidance. Risk assessment guidelines are required when safety concerns arise, such as when the pathogenicity of microorganisms increases or when GMOs are used in improperly controlled facilities. The guidelines state that most studies pose no or negligible risk and emphasize the importance of conducting a pragmatic evaluation based on evidence rather than overly complex regulation [12].

Improvements to the Synthetic Biology Development∙Experimentation System Subject to National Approval

In addition to the above-described current status survey and survey of related researchers, research on improving the approval system for the development and experimentation of LMOs related to synthetic biology has been conducted by an advisory committee consisting of synthetic biology experts and policy experts from academia, industry, national and public institutions, and public hearings. Based on this, we proposed a new avenue for improving national approval regulations for LMO development∙experimentation [9]. Among the four items subject to national approval, a proposal for the improvement of detailed regulations was proposed for two items which are closely related to synthetic biology research. (1) “In the case of using a microorganism whose species name is not specified and whose pathogenicity to humans is unknown” and (2) “Intentionally transferring drug resistance genes to microorganisms in a way that does not occur naturally” as shown in Table 5.

Table 5. Improvement plan for national approval system for LMO development and experimentation.

Subject	Current law	Improvement plan
Using a microorganism whose species name is not specified and whose pathogenicity to humans is unknown	Subject of national approval	Delete from the national approval list
Intentionally transferring drug resistance genes to microorganisms in a way that does not occur naturally	Approval exemption possible for 8 types of drug resistance genes	① Expansion of drug resistance genes subject to approval exemption	i) Addition of drug resistance genes, which are used as markers for gene manipulation with low human risk, to the genes subject to exemption from approval: high frequency of use (erythromycin, gentamicin, neomycin, spectinomycin), low frequency of use (blasticidin, sulfadiazine, nourseothricin, G418, aureobasidin)
	Approval exemption is limited to cases where 8 drug resistance genes are used in 7 approved host-vector systems	② Mitigation of approval subjects and procedures	ii) Approval exemption is proposed for introduction of the above drug resistance genes into RG1 microorganisms
			iii) In the case of introducing the above drug resistance genes into the RG2 microorganisms, it will be replaced with approval from the IBC. However, exceptions are made for Clostridium botulinum, Shigella dysenteriae Type 1, Chlamydia psittaci, and Vibrio cholerae O1∙O139, which are RG2 bacteria designated as high-risk pathogens. subject to approval

LMO=living modified organism; RG1=risk group 1; RG2=risk group 2; IBC=Institutional Biosafety Committee.

1. In the Case of Using a Microorganism Whose Species Name Is Not Specified and Whose Pathogenicity to Humans Is Unknown

For newly isolated microorganisms, it is often difficult to specify their exact species name because there is rarely a complete match when identifying strains using 16S rRNA sequencing. Currently, most strains used for LMO development are well-known and can be easily genetically manipulated. However, the development of LMOs targeting unknown microorganisms is very limited. Only 4% of the respondents to a survey of domestic synthetic biology researchers responded that they were conducting LMO development research on microorganisms of unknown species. However, with the development of synthetic biology, there is a possibility that LMO research using newly identified microorganisms with useful properties that are native to various environmental conditions will expand in the future. These regulations requiring national approval for the use of microorganisms whose species names and pathogenicity to humans are not specified are unique to Korean cases. Therefore, it would be beneficial to delete this rule from the items subject to national approval. However, if the definition of the concept is calibrated in the future or if the need for regulation arises, it can be reinstated.

2. Intentionally Transferring Drug Resistance Genes to Microorganisms in a Way That Does Not Occur Naturally

In 2017, the World Health Organization (WHO) published a list of 12 “priority pathogens” that pose the greatest threat to human health, classifying several microbes as Priority 1 consisting mainly of gram-negative pathogens with multidrug resistance to many antibiotics, including carbapenems and third-generation cephalosporins [13]. Pathogens resistant to one or two antibiotics that cause common diseases, such as gonorrhea, and food poisoning caused by Salmonella are in Priority 2 and 3. However, resistance genes for antibiotics, such as carbapenem, vancomycin, methicillin, clarithromycin, fluoroquinolone, and cephalosporin, to which pathogens belonging to Priority 1 and 2 are resistant, are not used as markers in LMO research. Ampicillin, which is included in Priority 3, is exempt from national approval. Therefore, non-pathogenic microorganisms are those in which the antibiotic resistance gene used in synthetic biology is introduced and does not cause infection or harm to the researcher conducting the experiment. In addition, approved LMO research facilities ensure the safe disposal of developed LMOs; therefore, the risk of leakage to the external environment is low and potential risks are not expected to be high, even in the case of leakage.

Concerns about the environmental release of antibiotic resistance genes by LMOs are the main reasons for the regulation of the use of antibiotic resistance genes in LMO research. However, there is no scientifically proven case for this. Indeed, there are an increasing number of microorganisms with drug resistance genes in the environment, but there is no scientific evidence that these genes have been released as a result of LMO studies. According to a study that analyzed antibiotic resistance genes in microorganisms in the upper, middle, and lower reaches of the Han River, the number of antibiotic-resistant microorganisms increased downstream, showing a correlation between human activity and increased resistance. This is interpreted as the fact that the fecal bacteria of humans or animals with natural resistance genes enter into the environment and spread to other bacteria via horizontal gene transfer [14]. Research findings suggest that antibiotic resistance genes found in the environment arise due to human activity, rather than being caused by the release of such genes from LMOs. Therefore, it has been proposed that antibiotic resistance genes, which have been proven safe for use as markers in synthetic biology research, should be exempt from national approval regulations. The proposed aim involves gradual deregulation to improve the efficiency of synthetic biology research and serves as a foundation for advanced biotechnology and biomanufacturing.

(1) The drug resistance genes currently used for LMO development should be included in the national approval exemption list.

(2) Classification of antibiotic resistance gene introduction in LMO research using non-pathogenic RG1 microorganisms as hosts for approval exemption. Current exemption-approved host vector systems do not include strains such as Corynebacterium glutamicum, which has already been industrially used and proven to be safe, making it difficult to view the current list as including all safe host vector systems. Although there may be a way to continuously increase the number of approved host-vector systems, it is reasonable to exempt drug resistance gene introduction studies targeting all RG1 strains that have been proven safe.

(3) The introduction of a drug resistance gene into RG2 microorganisms should be subject to IBC approval. Currently, only LMO studies targeting RG2, which do not involve drug resistance genes, are subject to IBC approval. However, considering the low potential risk to human safety when introducing drug resistance genes as markers, research on introducing drug resistance into RG2 microorganisms should also be subject to IBC approval. RG2 includes microorganisms that can cause relatively easy-to-prevent or treat diseases with mild symptoms in humans, such as the microbiome, which is a common human flora. Under current regulations, nationally approved research requires both IBC approval and national approval (Figure 1). Therefore, replacing national approval with IBC approval will promote research without the additional burden of IBC regulations. However, it may also require that efforts be made to strengthen the IBC capacity and management, particularly for small companies and schools. One solution is to use external IBCs if the formation of an in-house IBC is not feasible. Implementing these changes is warranted for the safety and proper management of LMO research facilities.

Conclusion

Due to the impact of synthetic biology on future industrial development, both securing the safety of LMO research and vitalizing related research, must be considered when establishing national approval regulations for the development and experimentation of synthetic biology-related LMO [15]. Since most of the LMOs developed through synthetic biology do not deviate from the scope of the existing LMO Act, it is necessary to create an environment in which researchers can legally conduct competitive research while complying with the regulations by improving the existing edicts [15]. The industrialization of LMOs requires a more thorough risk assessment, but the strict, superfluous regulations in the R&D stage act as major deterrents to the activation of related research. Therefore, rather than using the strategy of strict regulations considering every potential risk, realistic deregulation measures should be prepared first in consideration of the advancement of LMO research and requests from research sites. It will then be necessary to establish a system that can monitor related technology trends, continuously analyze risks, and prepare a scientific basis, to identify and supplement the appropriateness of regulations.

Supplementary Materials

Supplementary data is available online.