Appendix D
Biotechnology
For the purposes of this discussion, we consider “biotechnology” to refer to methods, products, and processes other than selective breeding and sexually crossing organisms to endow new characteristics in organisms. NASEM (2017) defines biotechnology products as
products developed through genetic engineering [(sometimes referred to as genome engineering)] (including products where the engineered DNA molecule is itself the “product,” as in an engineered molecule used as a DNA information-storage medium) or the targeted or in vitro manipulation of genetic information of organisms, including plants, animals, and microbes. The term also covers some products produced by such plants, animals, microbes, and cell-free systems or products derived from all of the above. (NASEM, 2017, p. 16)
Biotechnologies are anticipated to play a crucial role in biomanufacturing, which utilizes biological systems to produce commercially important biomolecules for use in the agricultural, food, material, energy, and pharmaceutical industries (Zhang et al., 2017). Recent federal initiatives in this area include the 2022 White House Executive Order 14081, Advancing Biotechnology and Biomanufacturing Innovation for a Sustainable, Safe, and Secure American Bioeconomy (Biden, 2022), the Defense Advanced Research Projects Agency’s (DARPA’s) Living Foundries program (Cheever, 2021), and the recent U.S. Department of Defense (DoD)-sponsored -sponsored BioMADE initiative.1 The DARPA Living Foundries program was developed to create a revolutionary, biologically based manufacturing platform to provide new materials, capabilities, and manufacturing paradigms for the DoD and the nation and seeks to develop the tools, technologies, and methodologies to transform biology into an engineering practice, speeding the biological design–build–test cycle and expanding the complexity of systems that can be engineered. In 2021, DoD sponsored BioMADE, a new manufacturing innovation institute to “enable domestic bioindustrial manufacturing at all scales, develop technologies to enhance U.S. bioindustrial competitiveness, de-risk investment in relevant infrastructure, and expand the biomanufacturing workforce to realize the economic promise of industrial biotechnology.”2
Increasingly, biotechnology applications are being designed to operate within the environment, some of which are also intended to spread or persist in those environments. These applications span agriculture, environmental remediation, climate mitigation, public health, and disease vector control. Some applications may fall within the U.S. Environmental Protection Agency’s (EPA’s) regulatory purview and at the same time could be used for EPA’s role in environmental protection, such as waste site remediation, creating a situation where a particular tool is both regulated and used by EPA.
REGULATION
EPA and the U.S. Department of Agriculture (USDA), and the U.S. Food and Drug Administration (FDA) have shared responsibility for regulating biotechnology in the United States. The 2017 Update to the Coordinated Framework indicates which agencies have responsibility for which types of biotechnology products (USEOP, 2017, p. 1). EPA is responsible for biotechnology products that have insecticidal, fungicidal, rodenticidal, or other toxic properties. The agency also has authority over new chemicals in
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1 See https://biomade.org.
2 See https://biomade.org.
commerce, which includes certain forms of genetically engineered organisms. The regulatory responsibilities include premarket testing and monitoring product performance.
Section 10402 of the CHIPS and Science Act of 2022 (P.L. 117-167) establishes a National Engineering Biology Research and Development Initiative to advance “engineering biology research; support risk research to address ethical, safety, security and other societal implications of engineering biology,” among other objectives. As part of the initiative, EPA is called on to “support research on how products, processes, and systems of engineering biology will affect or can protect the environment.” In addition, the 2022 White House Executive Order 14081, calls on EPA, USDA, and FDA to identify areas of ambiguity, gaps, or uncertainties in the Coordinated Framework by engaging with developers and external stakeholders, and through horizon scanning for novel products of biotechnology (Biden, 2022).
While many applications of biotechnology have a clear regulatory path through EPA, others raise questions as to which regulatory agency has the jurisdiction, as well as the proper scientific and public engagement expertise and resources to conduct risk assessments associated with such applications. As evident in the nearly 10-year debates in Key West, Florida, over the release of a genetically engineered mosquito, conducting a risk assessment without thorough public engagement can lead to public mistrust in the decision-making process for the approval of new tools or technologies (White, 2021).
Other new products will continue to pose challenges because the regulatory agencies do not have data points to which the new products can be easily compared. The potential use of gene drives provides one example.
A gene drive is a system of biasing inheritance to increase the likelihood of passing on a modified gene. Offspring inherit one copy of each gene from each parent. Normally, this limits the total incidence of mutations over generations. Gene-drive components cause the modified DNA to copy itself into the DNA from the unmodified parent. The result is a preferential increase in a specific trait from one generation to the next and, in time, possibly throughout the population. Gene drives have been suggested as a way to eliminate or reduce the transmission of disease, eradicate invasive species, or reverse pesticide resistance in agriculture (Gallo et al., 2018). The potential ability for gene drives to self-propagate has raised ecological and broader societal concerns, along with debates over how to govern such applications both domestically and internationally (Connolly et al., 2022; NASEM, 2016; Oye et al., 2014; Redford et al., 2019).
The development of gene drives has raised questions as to whether those funding the applications should also fund and develop guidelines for potential field trials; specifically, the National Institutes of Health (NIH, 2021) and DARPA. For example, in 2017, DARPA announced plans to award ~$65 million in contracts for its Safe Genes program. For comparison, in the face of its budgetary constraints (see Chapter 2), in July 2021, EPA’s Office of Research and Development (ORD) awarded $3,041,583 to five institutions to develop science-based approaches to evaluate the potential human health and environmental impacts of new biotechnology products.3 In addition, in May 2020, ORD issued a request for applications (RFA), as part of its Science to Achieve Results (STAR) program, for research funding to support the development of improved human health and environmental risk assessments of new biotechnology products, including chemicals; pesticides; and genetically modified microorganisms used in chemical production, microbial fuel cells, mining and resource extraction, building materials, waste remediation and pollution control, and nonpesticidal agriculture applications (e.g., biofertilizers, weather and climate modification; EPA, 2020).
IDENTIFICATION OF GENETICALLY MODIFIED MICROORGANISMS AND THEIR GENE PRODUCTS IN THE OPEN ENVIRONMENT
The design, mutation, introduction, and deletion of genes in developing genetically modified organisms (GMOs) requires an adduct to differentiate it from the parent or host strain, usually in the form of selectable antibiotic-resistant genes. For example, deletion of genetic elements on the chromosome may have kanamycin resistance associated with the modification, whereas introduction of plasmid-based
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3 See https://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/recipients.display/rfa_id/663#.
transformations will also have ampicillin resistance encoded. Concern arises if the GMO modification transfers to other microbiota in the environment, if the mutant strain overtakes the native population during the specific biotech application, or if the strain loses the introduced gene. Hence, there is a need to identify the genetic profile (host strain, modification, resistance gene) of an introduced, intact cell in the environment.
Molecular methods for detection of plasmids, gene products, and microbial populations are standard and routinely used. Quantitative polymerase chain reaction technology can be used to analyze the nucleic acid extracts from total, genomic, or plasmid DNA for quantifying gene copies or to analyze genetic variations in specific genomic regions and verify the presence of a modified gene. Sequencing the genes for16S ribosomal RNA (rRNA) provides species-level identification of microbial populations and thus the host strain. This technique is based on the detection of sequence differences in the highly variable regions of the 16S rRNA gene which is present in all bacteria.
Because microbes are abundant and varied in environmental samples, high-throughput sequencing, also referred to as next-generation sequencing (NGS), is used to determine the sequence of entire genomes or targeted regions of DNA or RNA by sequencing up to 100 billion reads of DNA fragments (150-300 base pairs) per run. This high-capacity approach is useful in metagenomics, in which the entire DNA extracted from an environmental sample is sequenced and aligned to a reference database, allowing gene identification and producing relative gene counts per sample. The totality of information generated by these methods can identify the presence of GMOs in a mixed soil or water sample, along with other bacteria and their genes.
However, there are analytical limitations to NGS. To verify that the mutation is associated with its host strain, selective antibiotic culturing of microorganisms from the sample is required, followed by 16S rDNA and plasmid or genomic sequencing. This time-consuming step reduces the number of isolates that can be processed. Furthermore, the read length of 16S sequencing limits the ability to make definitive taxonomic assignments during bioinformatics processing. To resolve base differences at the species or strain level, longer reads not offered by NGS of the 16S regions are needed.
Two approaches have been used to improve sequencing read length and whole-cell genomic contents. Single-molecule sequencing (Eid et al., 2009) can read up to several thousand base pairs of nucleotides directly from biological samples with high accuracy, but with fewer analytical steps than with traditional NGS. This technology was used to commercialize sequencing platforms and has many applications in biotechnology, microbial surveillance, and detection of genetic polymorphisms. The upgrade of NGS to longer sequence reads via single-molecule sequencing will vastly improve resolution and output.
Single-cell sequencing examines information from individual cells, providing a higher resolution of cellular differences than with traditional sequencing technology (Nawy, 2014; Shapiro et al., 2013; Tang et al., 2019; Wang and Navin, 2015). The technique has applications in health monitoring, genomics, and transcriptomics. However, methods to sequence a single strain’s 16S rDNA, chromosome, and plasmid have yet to be developed, but, when available, the methods will enable detection of GMOs in a mixed consortia collected from the environment.
ORD EXPERTISE
The expertise to make use of sequencing technologies exists within ORD. For example, ORD researchers have been using DNA metagenomic sequencing techniques to identify microorganisms in water as a means of identifying pathogens and other bacteria in environmental media (Annavajhalla et al., 2018). In addition, ORD researchers have recently published on the use of RNA sequencing as a means of screening biological responses to chemicals as a high-throughput method for predictive toxicology (Harrill et al., 2018). This expertise, coupled with informatics expertise, has already been applied in various aspects of prioritization, risk assessment, and regulation.
Maintaining expertise within EPA in emerging biotechnologies will be important. EPA has made some investments in this space. In 2012, EPA’s Office of Pollution Prevention and Toxics held a workshop in partnership with the Woodrow Wilson Center and the Massachusetts Institute of Technology Program
on Emerging Technologies. The workshop convened experts from academia, industry, government agencies, and nongovernmental organizations to develop a scientific understanding of the ecological issues that have the greatest relevance for evaluating synthetically designed organisms. Participants considered data needs and testing methods for assessing the safety of a field release of synthetically designed algae for biofuel production. EPA held a workshop in July 2021 to identify specific research needs related to the uncertain risks of emerging biotechnologies based on input from the EPA Office of Pollution Prevention and Toxics, Office of Pesticide Programs, ORD, and voluntary perspectives offered by Biotech STAR grant awardees and outside experts.
Other information-gathering activities could be useful for ORD to build on those previous workshops. Periodic horizon-scanning activities focused specifically on emerging biotechnologies are another valuable information-gathering activity, which could include research being conducted at other agencies as well as the private sector. A recent horizon scan (Kemp et al., 2020) identified 20 topics including the regulation of genomic data, increased philanthropic funding, malicious uses of neurochemicals, and environmental applications such as crops for changing climates and agricultural gene drives (see Table D-1).
TABLE D-1 Issues Identified Through a Bioengineering Horizon Scan in 2020
| <5 Years | 5–10 Years | >10 Years |
|---|---|---|
| Access to biotechnology through outsourcing | Agricultural gene drives | Bio-based production of materials |
| Crops for changing climates | Neuronal probes expanding new sensory capabilities | Live plant dispensers of chemical signals |
| Function-based design in protein engineering | Distributed pharmaceutical development and manufacturing | Malicious use of advanced neurochemistry |
| Philanthropy shapes bioscience research agendas | Genetically engineered phage therapy | Enhancing carbon sequestration |
| State and international regulation of DNA database use | Human genomics converging with computing technologies | Porcine bioengineered replacement organs |
| Microbiome engineering in agriculture | Governance of cognitive enhancement | |
| Phytoremediation of contaminated soils | ||
| Production of edible vaccines in plants | ||
| Rise of personalized medicine such as cell therapies |
NOTE: Issues are grouped according to likely timeline for realization.
SOURCE: Kemp et al., 2020. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0).
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