Self-sufficient primary natural killer cells engineered to express T cell receptors and interleukin-15 exhibit improved effector function and persistence.

Background: NK cells can be genetically engineered to express a transgenic T-cell receptor (TCR). This approach offers an alternative strategy to target heterogenous tumors, as NK:TCR cells can eradicate both tumor cells with high expression of HLA class I and antigen of interest or HLA class I negative tumors. Expansion and survival of NK cells relies on the presence of IL-15. Therefore, autonomous production of IL-15 by NK:TCR cells might improve functional persistence of NK cells. Here we present an optimized NK:TCR product harnessed with a construct encoding for soluble IL-15 (NK:TCR/IL-15), to support their proliferation, persistence and cytotoxic capabilities. Methods: Expression of tumor-specific TCRs in peripheral blood derived NK-cells was achieved following retroviral transduction. NK:TCR/IL-15 cells were compared with NK:TCR cells for autonomous cytokine production, proliferation and survival. NK:BOB1-TCR/IL-15 cells, expressing a HLA-B*07:02-restricted TCR against BOB1, a B-cell lineage specific transcription factor highly expressed in all B-cell malignancies, were compared with control NK:BOB1-TCR and NK:CMV-TCR/IL-15 cells for effector function against TCR antigen positive malignant B-cell lines in vitro and in vivo. Results: Viral incorporation of the interleukin-15 gene into engineered NK:TCR cells was feasible and high expression of the TCR was maintained, resulting in pure NK:TCR/IL-15 cell products generated from peripheral blood of multiple donors. Self-sufficient secretion of IL-15 by NK:TCR cells enables engineered NK cells to proliferate in vitro without addition of extra cytokines. NK:TCR/IL-15 demonstrated a marked enhancement of TCR-mediated cytotoxicity as well as enhanced NK-mediated cytotoxicity resulting in improved persistence and performance of NK:BOB1-TCR/IL-15 cells in an orthotopic multiple myeloma mouse model. However, in contrast to prolonged anti-tumor reactivity by NK:BOB1-TCR/IL-15, we observed in one of the experiments an accumulation of NK:BOB1-TCR/IL-15 cells in several organs of treated mice, leading to unexpected death 30 days post-NK infusion. Conclusion: This study showed that NK:TCR/IL-15 cells secrete low levels of IL-15 and can proliferate in an environment lacking cytokines. Repeated in vitro and in vivo experiments confirmed the effectiveness and target specificity of our product, in which addition of IL-15 supports TCR- and NK-mediated cytotoxicity.

High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation.

BACKGROUND: Methane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products. RESULTS: In this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high-cell-density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane. CONCLUSIONS: This study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.

Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin.

BACKGROUND: Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved. RESULTS: In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography-Mass spectrometry (UPLC-MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus. CONCLUSION: Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Sono-activatable engineered bacteria for antitumor therapy.

Bacteria show promising potential in tumor treatment, but safety concerns limit their application. In this issue, Gao et al.1 develop ultrasound-controlled engineered bacteria through integrating sono-activatable gene circuits, achieving local production and release of therapeutic cargos in tumor.

[Advances in base editing systems].

Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They have been widely used in biopharmaceutical research and crop engineering. Given the diverse needs for research and application, developing functional base editors has become a major focus in the field of genome editing. Currently, genome editing systems derived from clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR-Cas) and transcription activator-like effector (TALE) systems include single base editors, dual base editors, mitochondrial base editors, and CRISPR-related transposase systems. This review provides a comprehensive overview of the development of base editing systems, summarizes the characteristics, off-target effects, optimization, and improvement strategies of various base editors, and provides insights for further improvement and application of genome editing systems.

Strategies to Increase the Production of Triterpene Acids in Ligzhi or Reishi Medicinal Mushroom (Ganoderma lucidum, Agaricomycetes): A Review.

Ganoderic acids (GAs) are the main active ingredient of Ganoderma lucidum, which has been widely accepted as a medicinal mushroom. Due to the low yield of GAs produced by liquid cultured Ganoderma mycelium and solid cultured fruiting bodies, the commercial production and clinical application of GAs are limited. Therefore, it is important to increase the yield of GA in G. lucidum. A comprehensive literature search was performed with no set data range using the following keywords such as "triterpene," "ganoderic acids," "Ganoderma lucidum," and "Lingzhi" within the main databases including Web of Science, PubMed, and China National Knowledge Infrastructure (CNKI). The data were screened using titles and abstracts and those relevant to the topic were included in the paper and was not limited to studies published in English. Present review focuses on the four aspects: fermentation conditions and substrate, extrinsic elicitor, genetic engineering, and mutagenesis, which play significant roles in increasing triterpene acids production, thus providing an available reference for further research on G. lucidum fermentation.

Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

Synthetic bacteria designed using ars operons: a promising solution for arsenic biosensing and bioremediation.

The global concern over arsenic contamination in water due to its natural occurrence and human activities has led to the development of innovative solutions for its detection and remediation. Microbial metabolism and mobilization play crucial roles in the global cycle of arsenic. Many microbial arsenic-resistance systems, especially the ars operons, prevalent in bacterial plasmids and genomes, play vital roles in arsenic resistance and are utilized as templates for designing synthetic bacteria. This review novelty focuses on the use of these tailored bacteria, engineered with ars operons, for arsenic biosensing and bioremediation. We discuss the advantages and disadvantages of using synthetic bacteria in arsenic pollution treatment. We highlight the importance of genetic circuit design, reporter development, and chassis cell optimization to improve biosensors' performance. Bacterial arsenic resistances involving several processes, such as uptake, transformation, and methylation, engineered in customized bacteria have been summarized for arsenic bioaccumulation, detoxification, and biosorption. In this review, we present recent insights on the use of synthetic bacteria designed with ars operons for developing tailored bacteria for controlling arsenic pollution, offering a promising avenue for future research and application in environmental protection.

Antitumor activity of genetically engineered NK-cells in non-hematological solid tumor: a comprehensive review.

Recent advancements in genetic engineering have made it possible to modify Natural Killer (NK) cells to enhance their ability to fight against various cancers, including solid tumors. This comprehensive overview discusses the current status of genetically engineered chimeric antigen receptor NK-cell therapies and their potential for treating solid tumors. We explore the inherent characteristics of NK cells and their role in immune regulation and tumor surveillance. Moreover, we examine the strategies used to genetically engineer NK cells in terms of efficacy, safety profile, and potential clinical applications. Our investigation suggests CAR-NK cells can effectively target and regress non-hematological malignancies, demonstrating enhanced antitumor efficacy. This implies excellent promise for treating tumors using genetically modified NK cells. Notably, NK cells exhibit low graft versus host disease (GvHD) potential and rarely induce significant toxicities, making them an ideal platform for CAR engineering. The adoptive transfer of allogeneic NK cells into patients further emphasizes the versatility of NK cells for various applications. We also address challenges and limitations associated with the clinical translation of genetically engineered NK-cell therapies, such as off-target effects, immune escape mechanisms, and manufacturing scalability. We provide strategies to overcome these obstacles through combination therapies and delivery optimization. Overall, we believe this review contributes to advancing NK-cell-based immunotherapy as a promising approach for cancer treatment by elucidating the underlying mechanisms, evaluating preclinical and clinical evidence, and addressing remaining challenges.

Dual engineered bacteria improve inflammatory bowel disease in mice.

Currently, there are many different therapies available for inflammatory bowel disease (IBD), including engineered live bacterial therapeutics. However, most of these studies focus on producing a single therapeutic drug using individual bacteria, which may cause inefficacy. The use of dual drugs can enhance therapeutic effects. However, expressing multiple therapeutic drugs in one bacterial chassis increases the burden on the bacterium and hinders good secretion and expression. Therefore, a dual-bacterial, dual-drug expression system allows for the introduction of two probiotic chassis and enhances both therapeutic and probiotic effects. In this study, we constructed a dual bacterial system to simultaneously neutralize pro-inflammatory factors and enhance the anti-inflammatory pathway. These bacteria for therapy consist of Escherichia coli Nissle 1917 that expressed and secreted anti-TNF-α nanobody and IL-10, respectively. The oral administration of genetically engineered bacteria led to a decrease in inflammatory cell infiltration in colon and a reduction in the levels of pro-inflammatory cytokines. Additionally, the administration of engineered bacteria did not markedly aggravate gut fibrosis and had a moderating effect on intestinal microbes. This system proposes a dual-engineered bacterial drug combination treatment therapy for inflammatory bowel disease, which provides a new approach to intervene and treat IBD. KEY POINTS: • The paper discusses the effects of using dual engineered bacteria on IBD • Prospects of engineered bacteria in the clinical treatment of IBD.

AAV mediated genome engineering with a bypass coagulation factor alleviates the bleeding phenotype in a murine model of hemophilia B.

It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20-42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice.

Advances in hyaluronic acid production: Biosynthesis and genetic engineering strategies based on Streptococcus - A review.

Hyaluronic acid (HA), which is a highly versatile glycosaminoglycan, is widely applied across the fields of food, cosmetics, and pharmaceuticals. It is primary produced through Streptococcus fermentation, but the product presents inherent challenges concerning consistency and potential pathogenicity. However, recent strides in molecular biology have paved the way for genetic engineering, which facilitates the creation of high-yield, nonpathogenic strains adept at synthesizing HA with specific molecular weights. This comprehensive review extensively explores the molecular biology underpinning pivotal HA synthase genes, which elucidates the intricate mechanisms governing HA synthesis. Moreover, it delineates various strategies employed in engineering HA-producing strains.

Modelling daisy quorum drive: A short-term bridge across engineered fitness valleys.

Engineered gene-drive techniques for population modification and/or suppression have the potential for tackling complex challenges, including reducing the spread of diseases and invasive species. Gene-drive systems with low threshold frequencies for invasion, such as homing-based gene drive, require initially few transgenic individuals to spread and are therefore easy to introduce. The self-propelled behavior of such drives presents a double-edged sword, however, as the low threshold can allow transgenic elements to expand beyond a target population. By contrast, systems where a high threshold frequency must be reached before alleles can spread-above a fitness valley-are less susceptible to spillover but require introduction at a high frequency. We model a proposed drive system, called "daisy quorum drive," that transitions over time from a low-threshold daisy-chain system (involving homing-based gene drive such as CRISPR-Cas9) to a high-threshold fitness-valley system (requiring a high frequency-a "quorum"-to spread). The daisy-chain construct temporarily lowers the high thresholds required for spread of the fitness-valley construct, facilitating use in a wide variety of species that are challenging to breed and release in large numbers. Because elements in the daisy chain only drive subsequent elements in the chain and not themselves and also carry deleterious alleles ("drive load"), the daisy chain is expected to exhaust itself, removing all CRISPR elements and leaving only the high-threshold fitness-valley construct, whose spread is more spatially restricted. Developing and analyzing both discrete patch and continuous space models, we explore how various attributes of daisy quorum drive affect the chance of modifying local population characteristics and the risk that transgenic elements expand beyond a target area. We also briefly explore daisy quorum drive when population suppression is the goal. We find that daisy quorum drive can provide a promising bridge between gene-drive and fitness-valley constructs, allowing spread from a low frequency in the short term and better containment in the long term, without requiring repeated introductions or persistence of CRISPR elements.

Unveiling Methods to Stimulate Plant Resistance against Pathogens.

Plant diseases caused by pathogens pose significant threats to agricultural productivity and food security worldwide. The traditional approach of relying on chemical pesticides for disease management has proven to be unsustainable, emphasizing the urgent need for sustainable and environmentally friendly alternatives. One promising strategy is to enhance plant resistance against pathogens through various methods. This review aims to unveil and explore effective methods for stimulating plant resistance, transforming vulnerable plants into vigilant defenders against pathogens. We discuss both conventional and innovative approaches, including genetic engineering, induced systemic resistance (ISR), priming, and the use of natural compounds. Furthermore, we analyze the underlying mechanisms involved in these methods, highlighting their potential advantages and limitations. Through an understanding of these methods, scientists and agronomists can develop novel strategies to combat plant diseases effectively while minimizing the environmental impact. Ultimately, this research offers valuable insights into harnessing the plant's innate defense mechanisms and paves the way for sustainable disease management practices in agriculture.

Synthetic Genomics: Repurposing Biological Systems for Applications in Engineering Biology.

Substantial improvements in DNA sequencing and synthesis technologies and increased understanding of genome biology have empowered the development of synthetic genomics. The ability to design and construct engineered living cells boosted up by synthetic chromosomes provides opportunities to tackle enormous current and future challenges faced by humanity and the planet. Here we review the progresses, considerations, challenges, and future direction of the "design-build-test-learn" cycle used in synthetic genomics. We also discuss future applications enabled by synthetic genomics as this emerging field shapes and revolutionizes biomanufacturing and biomedicine.

Engineering the Signal Resolution of a Paper-Based Cell-Free Glutamine Biosensor with Genetic Engineering, Metabolic Engineering, and Process Optimization.

Specialized cancer treatments have the potential to exploit glutamine dependence to increase patient survival rates. Glutamine diagnostics capable of tracking a patient's response to treatment would enable a personalized treatment dosage to optimize the tradeoff between treatment success and dangerous side effects. Current clinical glutamine testing requires sophisticated and expensive lab-based tests, which are not broadly available on a frequent, individualized basis. To address the need for a low-cost, portable glutamine diagnostic, this work engineers a cell-free glutamine biosensor to overcome assay background and signal-to-noise limitations evident in previously reported studies. The findings from this work culminate in the development of a shelf-stable, paper-based, colorimetric glutamine test with a high signal strength and a high signal-to-background ratio for dramatically improved signal resolution. While the engineered glutamine test is important progress towards improving the management of cancer and other health conditions, this work also expands the assay development field of the promising cell-free biosensing platform, which can facilitate the low-cost detection of a broad variety of target molecules with high clinical value.

Persistence of genetically engineered canola populations in the U.S. and the adventitious presence of transgenes in the environment.

Feralization of genetically engineered (GE) crops increases the risk that transgenes will become integrated into natural and naturalizing plant populations. A key assumption of the management of GE crops is that populations of escaped plants are short-lived and therefore the risks they pose are limited. However, few populations of escaped crop plants have been tracked over the long term so our understanding of their persistence in ruderal or natural landscapes is limited. We repeated a large-scale road survey of feral GE canola populations in North Dakota, USA, initially conducted in 2010. Our objectives in 2021 were to determine the current distribution of feral canola populations, and to establish the relative frequency of GE and non-GE phenotypes in populations of canola throughout North Dakota. Our results indicate that, although the incidence of feral canola was less in 2021 than 2010, escaped canola populations remain common throughout the state. The prevalence of alternate forms of GE herbicide resistance changed between surveys, and we found an overabundance of non-GE plants compared to the frequency of non-transgenic forms in cultivation. Indirect evidence of persistence includes sampling plants with multiple transgenic traits, and finding populations far from transportation routes. We conclude that feral canola populations expressing transgenic herbicide resistance are established outside of cultivation, that they may be under selection for loss of the transgene, but that they nonetheless pose long-term risks by harboring transgenes in the unmanaged landscape.

Identifying widespread and recurrent variants of genetic parts to improve annotation of engineered DNA sequences.

Engineered plasmids have been workhorses of recombinant DNA technology for nearly half a century. Plasmids are used to clone DNA sequences encoding new genetic parts and to reprogram cells by combining these parts in new ways. Historically, many genetic parts on plasmids were copied and reused without routinely checking their DNA sequences. With the widespread use of high-throughput DNA sequencing technologies, we now know that plasmids often contain variants of common genetic parts that differ slightly from their canonical sequences. Because the exact provenance of a genetic part on a particular plasmid is usually unknown, it is difficult to determine whether these differences arose due to mutations during plasmid construction and propagation or due to intentional editing by researchers. In either case, it is important to understand how the sequence changes alter the properties of the genetic part. We analyzed the sequences of over 50,000 engineered plasmids using depositor metadata and a metric inspired by the natural language processing field. We detected 217 uncatalogued genetic part variants that were especially widespread or were likely the result of convergent evolution or engineering. Several of these uncatalogued variants are known mutants of plasmid origins of replication or antibiotic resistance genes that are missing from current annotation databases. However, most are uncharacterized, and 3/5 of the plasmids we analyzed contained at least one of the uncatalogued variants. Our results include a list of genetic parts to prioritize for refining engineered plasmid annotation pipelines, highlight widespread variants of parts that warrant further investigation to see whether they have altered characteristics, and suggest cases where unintentional evolution of plasmid parts may be affecting the reliability and reproducibility of science.