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.

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.

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.

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.

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.

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.

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.

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.

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.

What is the role of microbial biotechnology and genetic engineering in medicine?

Microbial products are essential for developing various therapeutic agents, including antibiotics, anticancer drugs, vaccines, and therapeutic enzymes. Genetic engineering techniques, functional genomics, and synthetic biology unlock previously uncharacterized natural products. This review highlights major advances in microbial biotechnology, focusing on gene-based technologies for medical applications.

Two-Photon FRET/FLIM Imaging of Cerebral Neurons.

Two-photon FRET (Förster resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) enable the detection of FRET changes of fluorescence reporters in deep brain tissues, which provide a valuable approach for monitoring target molecular dynamics and functions. Here, we describe two-photon FRET and FLIM imaging techniques that allow us to visualize endogenous and optogenetically induced cAMP dynamics in living neurons with genetically engineered FRET-based cAMP reporters.

Current approaches to genetic modification of marine bacteria and considerations for improved transformation efficiency.

Marine bacteria play vital roles in symbiosis, biogeochemical cycles and produce novel bioactive compounds and enzymes of interest for the pharmaceutical, biofuel and biotechnology industries. At present, investigations into marine bacterial functions and their products are primarily based on phenotypic observations, -omic type approaches and heterologous gene expression. To advance our understanding of marine bacteria and harness their full potential for industry application, it is critical that we have the appropriate tools and resources to genetically manipulate them in situ. However, current genetic tools that are largely designed for model organisms such as E. coli, produce low transformation efficiencies or have no transfer ability in marine bacteria. To improve genetic manipulation applications for marine bacteria, we need to improve transformation methods such as conjugation and electroporation in addition to identifying more marine broad host range plasmids. In this review, we aim to outline the reported methods of transformation for marine bacteria and discuss the considerations for each approach in the context of improving efficiency. In addition, we further discuss marine plasmids and future research areas including CRISPR tools and their potential applications for marine bacteria.

The design of an RGD in situ sustained delivery system utilizing scallop byssal protein through genetic engineering.

Although bioactive peptides enhancing bone healing have demonstrated effectiveness in treating bone defects, in vivo instability poses a challenge to their clinical application. Currently reported peptide delivery systems do not meet the demands of bone tissue repair regarding stability and peptide release efficacy. Herein, the self-assembling recombinant chimeric protein (Sbp5-2RGD) is developed by genetic engineering with cell adhesion peptide RGD as the targeted peptide and a newly discovered scallop byssal-derived protein Sbp5-2 that can assemble into wet stable films as the structural domain. In vitro studies show that the Sbp5-2RGD film exhibits excellent extensibility and biocompatibility. In vitro and in vivo degradation experiments demonstrate that the film remains stable due to the layer-by-layer degradation mode, resulting in sustained delivery of RGD in situ for up to 4 weeks. Consequently, the film can effectively promote osteogenesis, which accelerates bone defect healing and the implants osseointegration. Cell-level studies further show that the film up-regulates the expression of genes and proteins (ALP, OCN, OSX, OPN, RUNX2, VEGF) associated with osteogenesis and angiogenesis. Overall, this novel protein film represents an intelligent platform for peptide immobilization, protection, and release through its self-assembly, dense structure, and degradation mode, providing a therapeutic strategy for bone repair.

Genetically engineered cell-derived nanovesicles for cancer immunotherapy.

The emergence of immunotherapy has marked a new epoch in cancer treatment, presenting substantial clinical benefits. Extracellular vesicles (EVs), as natural nanocarriers, can deliver biologically active agents in cancer therapy with their inherent biocompatibility and negligible immunogenicity. However, natural EVs have limitations such as inadequate targeting capability, low loading efficacy, and unpredictable side effects. Through progress in genetic engineering, EVs have been modified for enhanced delivery of immunomodulatory agents and antigen presentation with specific cancer targeting ability, deepening the role of EVs in cancer immunotherapy. This review briefly describes typical EV sources, isolation methods, and adjustable targeting of EVs. Furthermore, this review highlights the genetic engineering strategies developed for delivering immunomodulatory agents and antigen presentation in EV-based systems. The prospects and challenges of genetically engineered EVs as cancer immunotherapy in clinical translation are also discussed.

Application of multiple strategies to enhance oleanolic acid biosynthesis by engineered Saccharomyces cerevisiae.

Oleanolic acid and its derivatives are widely used in the pharmaceutical, agricultural, cosmetic and food industries. Previous studies have shown that oleanolic acid production levels in engineered cell factories are low, which is why oleanolic acid is still widely extracted from traditional medicinal plants. To construct a highly efficient oleanolic acid production strain, rate-limiting steps were regulated by inducible promoters and the expression of key genes in the oleanolic acid synthetic pathway was enhanced. Subsequently, precursor pool expansion, pathway refactoring and diploid construction were considered to harmonize cell growth and oleanolic acid production. The multi-strategy combination promoted oleanolic acid production of up to 4.07 g/L in a 100 L bioreactor, which was the highest level reported.

ORBIT for E. coli: kilobase-scale oligonucleotide recombineering at high throughput and high efficiency.

Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for Escherichia coli. Our redesigned plasmid toolkit for oligonucleotide recombineering achieved significantly higher efficiency than λ Red double-stranded DNA recombineering and enabled precise, stable knockouts (≤134 kb) and integrations (≤11 kb) of various sizes. Additionally, we constructed multi-mutants in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.

Perspectives on Genetically Engineered Microorganisms and Their Regulation in the United States.

Genetically engineered microorganisms (GEMs) represent a new paradigm in our ability to address the needs of a growing, changing world. GEMs are being used in agriculture, food production and additives, manufacturing, commodity and noncommodity products, environmental remediation, etc., with even more applications in the pipeline. Along with modern advances in genome-manipulating technologies, new manufacturing processes, markets, and attitudes are driving a boom in more products that contain or are derived from GEMs. Consequentially, researchers and developers are poised to interact with biotechnology regulatory policies that have been in effect for decades, but which are out of pace with rapidly changing scientific advances and knowledge. In the United States, biotechnology is regulated by multiple agencies with overlapping responsibilities. This poses a challenge for both developers and regulators to simultaneously allow new innovation and products into the market while also ensuring their safety and efficacy for the public and environment. This article attempts to highlight the various factors that interact between regulatory policy and development of GEMs in the United States, with perspectives from both regulators and developers. We present insights from a 2022 workshop hosted at the University of California, Berkeley that convened regulators from U.S. regulatory agencies and industry developers of various GEMs and GEM-derived products. We highlight several new biotechnologies and applications that are driving innovation in this space, and how regulatory agencies evaluate and assess these products according to current policies. Additionally, we describe recent updates to regulations that incorporate new technology and knowledge and how they can adapt further to effectively continue regulating for the future.

Diffusion-Based Generative Network for de Novo Synthetic Promoter Design.

Computer-aided promoter design is a major development trend in synthetic promoter engineering. Various deep learning models have been used to evaluate or screen synthetic promoters, but there have been few works on de novo promoter design. To explore the potential ability of generative models in promoter design, we established a diffusion-based generative model for promoter design in Escherichia coli. The model was completely driven by sequence data and could study the essential characteristics of natural promoters, thus generating synthetic promoters similar to natural promoters in structure and component. We also improved the calculation method of FID indicator, using a convolution layer to extract the feature matrix of the promoter sequence instead. As a result, we got an FID equal to 1.37, which meant synthetic promoters have a distribution similar to that of natural ones. Our work provides a fresh approach to de novo promoter design, indicating that a completely data-driven generative model is feasible for promoter design.

Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.