Environmental risk assessment of advanced therapies containing genetically modified organisms in the EU.

Gene therapy medicinal products have the potential to provide curative treatment for many diseases with current limited therapeutic options. As advanced therapy medicinal products (ATMPs), these therapies undergo a centralised, single European Union authorisation by the European Medicines Agency (EMA), but the risks and potential harm to the environment and population at large are weighted in each application, and different interpretations at national level exist. A streamlined procedure is now in place to facilitate a consistent approach for the assessment of the environmental risks of medicines containing genetically modified organisms for both clinical trial applications and marketing authorisation applications. This article provides an overview of basic requirements across the EU, an overview of the new streamlined process and discusses available guidance for developers with particular emphasis on marketing authorisation applications. All these initiatives are aimed to remove hurdles for ATMP developers and facilitate faster access to patients.

Gene Drive-Modified Organisms: Developing Practical Risk Assessment Guidance.

Risk assessors, risk managers, developers, potential applicants, and other stakeholders at many levels discuss the need for new or further risk assessment guidance for deliberate environmental releases of gene drive-modified organisms. However, preparing useful and practical guidance entails challenges, to which we offer recommendations based on our experience drafting guidance.

Building biosecurity for synthetic biology.

The fast-paced field of synthetic biology is fundamentally changing the global biosecurity framework. Current biosecurity regulations and strategies are based on previous governance paradigms for pathogen-oriented security, recombinant DNA research, and broader concerns related to genetically modified organisms (GMOs). Many scholarly discussions and biosecurity practitioners are therefore concerned that synthetic biology outpaces established biosafety and biosecurity measures to prevent deliberate and malicious or inadvertent and accidental misuse of synthetic biology's processes or products. This commentary proposes three strategies to improve biosecurity: Security must be treated as an investment in the future applicability of the technology; social scientists and policy makers should be engaged early in technology development and forecasting; and coordination among global stakeholders is necessary to ensure acceptable levels of risk.

Spatiotemporal Controllability and Environmental Risk Assessment of Genetically Engineered Gene Drive Organisms from the Perspective of European Union Genetically Modified Organism Regulation.

Gene drive organisms are a recent development created by using methods of genetic engineering; they inherit genetic constructs that are passed on to future generations with a higher probability than with Mendelian inheritance. There are some specific challenges inherent to the environmental risk assessment (ERA) of genetically engineered (GE) gene drive organisms because subsequent generations of these GE organisms might show effects that were not observed or intended in the former generations. Unintended effects can emerge from interaction of the gene drive construct with the heterogeneous genetic background of natural populations and/or be triggered by changing environmental conditions. This is especially relevant in the case of gene drives with invasive characteristics and typically takes dozens of generations to render the desired effect. Under these circumstances, "next generation effects" can substantially increase the spatial and temporal complexity associated with a high level of uncertainty in ERA. To deal with these problems, we suggest the introduction of a new additional step in the ERA of GE gene drive organisms that takes 3 criteria into account: the biology of the target organisms, their naturally occurring interactions with the environment (biotic and abiotic), and their intended biological characteristics introduced by genetic engineering. These 3 criteria are merged to form an additional step in ERA, combining specific "knowns" and integrating areas of "known unknowns" and uncertainties, with the aim of assessing the spatiotemporal controllability of GE gene drive organisms. The establishment of assessing spatiotemporal controllability can be used to define so-called "cut-off criteria" in the risk analysis of GE gene drive organisms: If it is likely that GE gene drive organisms escape spatiotemporal controllability, the risk assessment cannot be sufficiently reliable because it is not conclusive. Under such circumstances, the environmental release of the GE gene drive organisms would not be compatible with the precautionary principle (PP). Integr Environ Assess Manag 2020;16:555-568. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Options to Reform the European Union Legislation on GMOs: Risk Governance.

Here, we discuss options to reform the EU genetically modified organism (GMO) regulatory framework, to make risk assessment and decision-making more consistent with scientific principles, and to lay the groundwork for international coherence. We discussed the scope and definitions in a previous article and, thus, here we focus on the procedures for risk assessment and risk management.

Modulation of culture medium confers high-specificity production of isopentenol in Bacillus subtilis.

Enthusiasm for mining isoprenoid-based flavors, pharmaceuticals, and nutraceuticals from GRAS (Generally Regarded as Safe) status microbial hosts has increased in the past few years due to the limitations associated with their plant-based extraction and chemical synthesis. Bacillus subtilis, a well-known GRAS microbe, is a promising alternative due to its fast growth rate and the ability to metabolize complex carbon sources. The study focused on the high-specificity production of isopentenol in B. subtilis by modulating the culture medium. Media modulation led to a 2.5 folds improvement in isopentenol titer in the wild-type strain. In the recombinant strain, optimization of physico-chemical factors, coupled with overexpression of the nudF enzyme resulted in a maximum isopentenol titer of ∼6 mg/L in a shake flask. The recombinant strain produced ∼5 mg/L isoprenol (∼80% of the total isopentenol production) and ∼1.8 mg/L prenol (∼65% of the total isopentenol production) by utilizing sorbitol and pyruvate as the carbon sources, respectively. Replacement of glucose with sorbitol and pyruvate reduced the production of the undesired metabolites and enhanced high-specificity production of isopentenol. Upon replacement of the carbon source with a low-cost substrate, a non-detoxified rice-straw hydrolysate, the engineered strain produced 2.19 mg/L isopentenol. This proof-of-concept study paves the path for the high-specificity production and cost-effective recovery of isopentenol from industrially competent microbial strains with engineered isoprenoid pathways.

Mitigation of environmental pollution by genetically engineered bacteria - Current challenges and future perspectives.

Industries are the paramount driving force for the economic and technological development of society. However, the flourishing industrialization and unimpeded growth of current production unit's result in widespread environmental pollution due to increased discharge of wastes loaded with baleful, hazardous, and carcinogenic contaminants. Physicochemical-based remediation means are costly, create a secondary disposal problem and remain inadequate for pollution mitigating because of the continuous emergence of new recalcitrant pollutants. Due to eco-friendly, social acceptance, and lesser health hazards, microbial bioremediation has received considerable global attention for pollution abatement. Moreover, with the recent advancement in biotechnology and microbiology, genetically engineered bacteria with high ability to remove environmental pollutants are widely used in the fields of environmental restoration, resulting in the bioremediation in a more viable and eco-friendly way. This review summarized the advantages of genetically engineered bacteria and their application in the treatment of a wide variety of environmental contaminants such as synthetic dyestuff, heavy metal, petroleum hydrocarbons, polychlorinated biphenyls, phenazines and agricultural chemicals which will include herbicides, pesticides, and fertilizers. Considering the risk of genetic material exchange by using genetically engineered bacteria, the challenges and limitations associated with the application of recombinant bacteria on contaminated sites are also discussed. An integrated microbiological, biological and ecological acquaintance accompanied by field engineering designs are the desired features for effective in situ bioremediation of hazardous waste polluted sites by recombinant bacteria.

Perspective: regulation of pest and disease control strategies and why (many) economists are concerned.

Pests and diseases are a continuous challenge in agriculture production. A wide range of control strategies have been and will continue to be developed. New control strategies are in almost all countries around the world assessed prior to approval for use in farmers' fields. This is rightly so to avoid and even reduce negative effects for human health and the environment. Over the past decades the approval processes have become increasingly politicized resulting in an increase in the direct approval costs and the length in approval time without increasing the safety of the final product. This reduces the development of control strategies and often has negative human health and environmental effects. Possibilities exist for improvements. They include reducing approval costs and approval time by streamlining the approval process and substituting approval requirements by strengthening ex-post liability. © 2018 Society of Chemical Industry.

Not Just Asking Questions: Effects of Implicit and Explicit Conspiracy Information About Vaccines and Genetic Modification.

While conspiracy ideation has attracted overdue attention from social scientists in recent years, little work focuses on how different pro-conspiracy messages affect the take-up of conspiracy beliefs. In this study, we compare the effect of explicit and implicit conspiracy cues on the adoption of conspiracy beliefs. We also examine whether corrective information can undo conspiracy cues, and whether there are differences in the effectiveness of corrective information based on whether a respondent received an explicit or implicit conspiracy cue. We examine these questions using a real-world but low-salience conspiracy theory concerning Zika, GM mosquitoes, and vaccines. Using a preregistered experiment (N = 1018: https://osf.io/hj2pw/), we find that both explicit and implicit conspiracy cues increase conspiracy beliefs, but in both cases corrections are generally effective. We also find reception of an explicit conspiracy cue and its correction is conditional on feelings toward the media and pharmaceutical companies. Finally, we find that examining open-ended conspiracy belief items reveals similar patterns, but with a few key differences. These findings have implications for how news media cover controversial public health issues going forward.

Function of three RuBisCO enzymes under different CO2 conditions in Hydrogenovibrio marinus.

The obligate chemolithoautotrophic bacterium, Hydrogenovibrio marinus MH-110 has three ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isoenzymes, designated CbbLS-1, CbbLS-2, and CbbM, which are encoded by the cbbL1S1, cbbL2S2, and cbbM genes, respectively. Functions of these isoenzymes at different CO2 concentrations were investigated using deletion mutants of their genes. Deletion of cbbL1 had no effect on cell growth under any of the test growth conditions. The cbbL2 mutant was unable to grow under lower (≤0.15%) CO2 conditions, though it grew normally under higher (≥2%) CO2 conditions. Growth of the cbbM mutant was retarded under higher CO2 conditions but was not affected by lower CO2 conditions. These results indicate that CbbLS-2 and CbbM specifically function under lower and higher CO2 conditions, respectively. The growth retardation of the cbbL2 and cbbM mutants was not restored by complementation with plasmids carrying the cbbL2S2 and cbbM genes, respectively. The cbbL2S2 and cbbM genes are followed by the carboxysome genes and the cbbQmOm genes, respectively. Co-expression of these downstream genes was probably necessary for the in vivo function of CbbLS-2 and CbbM. CbbLS-1 was upregulated in the cbbL2 and cbbM mutants under the lower and higher CO2 conditions, respectively, indicating that the expression of cbbL1S1 was controlled to compensate the deficiency of the other RuBisCO isoenzymes.

Considerations for the governance of gene drive organisms.

Governance is a broader and more flexible concept than statute-driven regulations as it incorporates components outside the latter's remit. Considerations of governance are critical in the development of emerging biotechnologies such as gene drive organisms. These have been proposed or are being developed to address public and environmental health issues not addressed easily by conventional means. Here, we consider how the concept of governance differs from statute-driven regulation with reference to the role each may play in the development of gene drive organisms. First, we discuss existing statute-based regulatory systems. Second, we consider whether novel risks or different concerns derive from gene drive organisms, concentrating on characteristics that contribute to public health or environmental risk and uncertainties that may affect risk perceptions. Third, we consider public engagement, outlining how existing statute-driven regulatory systems and other governance mechanisms may provide opportunities for constructive interactions. Finally, we provide some observations that may help address science- and values-based concerns in a governance space larger than that of statute-driven regulatory systems.

Circulating cell-free DNA as a biomarker of tissue injury: Assessment in a cardiac xenotransplantation model.

BACKGROUND: Observational studies suggest that cell-free DNA (cfDNA) is a biomarker of tissue injury in a range of conditions including organ transplantation. However, the lack of model systems to study cfDNA and its relevance to tissue injury has limited the advancements in this field. We hypothesized that the predictable course of acute humoral xenograft rejection (AHXR) in organ transplants from genetically engineered donors provides an ideal system for assessing circulating cfDNA as a marker of tissue injury. METHODS: Genetically modified pig donor hearts were heterotopically transplanted into baboons (n = 7). Cell-free DNA was extracted from pre-transplant and post-transplant baboon plasma samples for shotgun sequencing. After alignment of sequence reads to pig and baboon reference sequences, we computed the percentage of xenograft-derived cfDNA (xdcfDNA) relative to recipient by counting uniquely aligned pig and baboon sequence reads. RESULTS: The xdcfDNA percentage was high early post-transplantation and decayed exponentially to low stable levels (baseline); the decay half-life was 3.0 days. Post-transplantation baseline xdcfDNA levels were higher for transplant recipients that subsequently developed graft loss than in the 1 animal that did not reject the graft (3.2% vs 0.5%). Elevations in xdcfDNA percentage coincided with increased troponin and clinical evidence of rejection. Importantly, elevations in xdcfDNA percentage preceded clinical signs of rejection or increases in troponin levels. CONCLUSION: Cross-species xdcfDNA kinetics in relation to acute rejection are similar to the patterns in human allografts. These observations in a xenotransplantation model support the body of evidence suggesting that circulating cfDNA is a marker of tissue injury.

Bacterial Bioreporter-Based Mercury and Phenanthrene Assessment in Yangtze River Delta Soils of China.

Genetically engineered bacterial whole-cell bioreporters were deployed to investigate bioavailable mercury (b-Hg) and phenanthrene (b-PHE). Characterized by high sensitivity and specificity in aqueous solutions, the bioreporter system could detect in amended soils the concentrations of b-Hg and b-PHE in the ranges of 19.6 to 111.6 and 21.5 to 110.9 µg kg, respectively. The sensitivity of the system allowed for the combined analysis of b-Hg and b-PHE from real environmental samples. Therefore, soil samples from three large refinery facilities were tested, and the results from the instrumental analysis strongly correlated with the ones obtained with the bioreporter method. Large-scale and fast screening of soil contamination across the Yangtze River Delta in Eastern China was conducted. More than 36% of the samples contained b-Hg, whereas the fractions of b-PHE were below the detection limit for all the samples. These results indicated a higher toxicity and more hazardous condition for Hg contamination than for PHE. Population densities and airborne 10-µm particulate matter (PM10) concentrations were used as parameters for comparison with the spatial distribution of the b-Hg and b-PHE fractions. The results revealed that the bioreporters could offer a rapid and cost-efficient method to test soil samples from contaminated areas and provide a screening tool for environmental risk assessment.

Using a Time-Driven Activity-Based Costing Model To Determine the Actual Cost of Services Provided by a Transgenic Core.

Laboratory animal programs and core laboratories often set service rates based on cost estimates. However, actual costs may be unknown, and service rates may not reflect the actual cost of services. Accurately evaluating the actual costs of services can be challenging and time-consuming. We used a time-driven activity-based costing (ABC) model to determine the cost of services provided by a resource laboratory at our institution. The time-driven approach is a more efficient approach to calculating costs than using a traditional ABC model. We calculated only 2 parameters: the time required to perform an activity and the unit cost of the activity based on employee cost. This method allowed us to rapidly and accurately calculate the actual cost of services provided, including microinjection of a DNA construct, microinjection of embryonic stem cells, embryo transfer, and in vitro fertilization. We successfully implemented a time-driven ABC model to evaluate the cost of these services and the capacity of labor used to deliver them. We determined how actual costs compared with current service rates. In addition, we determined that the labor supplied to conduct all services (10,645 min/wk) exceeded the practical labor capacity (8400 min/wk), indicating that the laboratory team was highly efficient and that additional labor capacity was needed to prevent overloading of the current team. Importantly, this time-driven ABC approach allowed us to establish a baseline model that can easily be updated to reflect operational changes or changes in labor costs. We demonstrated that a time-driven ABC model is a powerful management tool that can be applied to other core facilities as well as to entire animal programs, providing valuable information that can be used to set rates based on the actual cost of services and to improve operating efficiency.

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms.

The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing. Graphical abstract There are generally three different options for absolute quantification of genetically modified organisms (GMOs) using digital PCR: droplet- or chamber-based and droplets in chambers. All have in common the distribution of reaction mixture into several partitions, which are all subjected to PCR and scored at the end-point as positive or negative. Based on these results GMO content can be calculated.

Current (Food) Allergenic Risk Assessment: Is It Fit for Novel Foods? Status Quo and Identification of Gaps.

Food allergies are recognized as a global health concern. In order to protect allergic consumers from severe symptoms, allergenic risk assessment for well-known foods and foods containing genetically modified ingredients is installed. However, population is steadily growing and there is a rising need to provide adequate protein-based foods, including novel sources, not yet used for human consumption. In this context safety issues such as a potential increased allergenic risk need to be assessed before marketing novel food sources. Therefore, the established allergenic risk assessment for genetically modified organisms needs to be re-evaluated for its applicability for risk assessment of novel food proteins. Two different scenarios of allergic sensitization have to be assessed. The first scenario is the presence of already known allergenic structures in novel foods. For this, a comparative assessment can be performed and the range of cross-reactivity can be explored, while in the second scenario allergic reactions are observed toward so far novel allergenic structures and no reference material is available. This review summarizes the current analytical methods for allergenic risk assessment, highlighting the strengths and limitations of each method and discussing the gaps in this assessment that need to be addressed in the near future.