Genetic Engineering Technology Research Articles

Advances of monoclonal antibodies and analysis of marketed antibody drugs

In recent years, there has been a frequent occurrence of various epidemics worldwide such as COVID-19, monkeypox, influenza, and others additionally, there has been an increase in the number of new patients diagnosed with various types of tumors. Traditional drugs have limited effectiveness against emerging infectious diseases, tumors, and autoimmune diseases. However, with the emergence of hybridoma technology, monoclonal antibodies have achieved extensive applications and antibody drugs are playing an important role in modern medicine. Monoclonal antibodies have undergone various development stages, starting from mouse-derived antibodies to human-mouse chimeric antibodies, humanized antibodies, and ultimately human antibodies. Throughout this process, their immunogenicity has gradually decreased, while their safety for human use steadily increased. Fully human antibodies are currently the safest form of antibody, because their sequences all come from human sources and they do not induce human anti-murine antibody reactions. With the advance of genetic engineering technology, flow cytometry coupled to single B cell gene amplification technology has made it easier to construct and screen for fully human monoclonal antibodies. The development of antibody drugs has provided new opportunities, and the market for monoclonal antibody drugs will further expand. This article reviews the research progress of monoclonal antibodies and presents information on the 163 monoclonal antibody drugs approved by the United States Food and Drug Administration (FDA) as of Oct 1st, 2023. The aim is to offer new insights for the development and production of monoclonal antibodies in China.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function

BackgroundMycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown.MethodsMtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni–NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs.ResultsIn this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αβ T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb.ConclusionActivated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

115 Gene editing: Where are we on campus and where can it go?

Abstract Editing the genome of livestock provides solutions to otherwise intractable problems and can provide a tool to better understand the role of specific proteins during development. The potential benefit for production agriculture is so great that the USDA Agricultural Research Service has invested in 4 positions now located in a research unit at the University of Missouri with a focus currently on swine and poultry. Genome editing can result in introducing alleles from divergent populations or novel alleles to produce animals that have new proteins, modified proteins or animals that no longer produce a protein. Applications of genome editing include improving disease resistance (viral and bacterial), improving digestion, disseminating valuable genetics, improved thermoregulation, changing the body/milk composition, and creating hypoallergenic meat. Changes in these animals will improve animal welfare, animal productivity and the sustainability of animal agriculture. In addition to improving animal agriculture, gene editing has enabled a new understanding of maternal recognition of pregnancy in the pig and a better understanding of molecules involved in growth and development of pigs. With new developments in editing of just somatic cells [somatic cell gene editing (SCGE), rather than germline editing], it will be possible to edit some cells in existing animals and thus change their phenotype. SCGE technologies such as this are being developed via the biomedical community as models to lead to human clinical applications. One such application is to correct cells in the lungs that produce the CFTR protein. Correction of just 10% of the cells in the lungs is estimated to correct the lung symptoms associated with cystic fibrosis. Similar applications in production agriculture might produce animals that have novel phenotypes of an altered carcass composition or better use of nutrients. Application of genetic engineering technologies such as genome editing is limited by biology and the imagination of the investigator.

Genetically Engineered CLDN18.2 CAR-T Cells Expressing Synthetic PD1/CD28 Fusion Receptors Produced Using a Lentiviral Vector.

This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer.

Platelet Membrane-Coated r-SAK Improves Thrombolytic Efficacy by Targeting Thrombus.

Thrombolytic therapy is one of the most effective treatments for thrombus dissolution and recanalization of blocked vessels in thrombotic diseases. However, the application of the thrombolytic strategy has been limited due to unsatisfactory thrombolytic efficacy, relatively higher bleeding complications, and consequently restricted indications. Recombinant staphylokinase (r-SAK) is a third-generation thrombolytic agent produced by genetic engineering technology, which exhibits a better thrombolytic efficacy than urokinase and recombinant streptokinase. Inspired by the natural affinity of platelets in hemostasis and pathological thrombosis, we developed a platelet membrane (PM)-coated r-SAK (PM-r-SAK). Results from animal experiments and human in vitro studies showed that the PM-r-SAK had a thrombolytic efficacy equal to or better than its 4-fold dose of r-SAK. In a totally occluded rabbit femoral artery thrombosis model, the PM-r-SAK significantly shortened the initial recanalization time compared to the same dose and 4-fold dose of r-SAK. Regarding the recanalized vessels, the PM-r-SAK prolonged the time of reperfusion compared to the same dose and 4-fold dose of r-SAK, though the differences were not significant. An in vitro thrombolytic experiment demonstrated that the thrombolytic efficacy of PM-r-SAK could be inhibited by platelet-poor plasma from patients taking aspirin and ticagrelor. PM coating significantly improves the thrombolytic efficacy of r-SAK, which is related to the thrombus-targeting activity of the PM-r-SAK and can be inhibited by aspirin- and ticagrelor-treated plasma.

Competencies and training needs of extension agents for educating farmers on genetically engineered crops in Uganda

The purpose of this study was to determine the training needs of extension agents in Uganda to lead successful education programs on genetically engineered (GE) crops. This was a descriptive survey research study conducted online with public agricultural extension agents in the eastern agro-ecological zone of Uganda. This study used Borich’s method to identify training needs. A survey instrument was designed to determine extension agents’ perceived importance and proficiency of 60 competencies organized under the eight Public Issues Education (PIE) framework competency constructs. The survey received 58 usable responses comprising an 83% response rate. All eight PIE competency constructs were perceived by the extension agents to be important. This study identified additional four competencies important for PIE in addition to the eight competencies in the model. Agents’ greatest training needs were creating partnerships and designing GE education programs. The lowest training needs were creating an environment of professionalism and managing conflicts. The findings indicate the importance of training extension agents on how to engage with farmers in new ways to educate them on GE technology. This study provides implications for determining the training needs of extension agents in PIE such as educating farmers on GE technology.

It takes two to tango with CRISPR: a history and overview of augmenting the technology for genetic engineering

It takes two to tango with CRISPR: a history and overview of augmenting the technology for genetic engineering

Cardiac Amyloidosis: A Contemporary Review of Medical and Surgical Therapy.

Amyloidosis is a systemic disease initiated by deposition of misfolded proteins in the extracellular space, due to which multiple organs may be affected concomitantly. Cardiac amyloidosis, however, remains a major cause of morbidity and mortality in this population due to infiltrative /restrictive cardiomyopathy. This review attempts to focus on contemporary medical and surgical therapies for the different types of cardiac amyloidosis. Amyloidosis affecting the heart are predominantly of the transthyretin type (acquired in the older or genetic in the younger patients), and the monoclonal immunoglobulin light chain (AL) type which is solely acquired. A rare form of secondary amyloidosis AA type can also affect the heart due to excessive production and accumulation of the acute-phase protein called Serum Amyloid A" (SAA) in the setting of chronic inflammation, cancers or autoinflammatory disease. More commonly AA amyloidosis is seen in the liver and kidney. Other rare types are Apo A1 and Isolated Atrial Amyloidosis (AANF). Medical therapies have made important strides in the clinical management of the two common types of cardiac amyloidosis. Surgical therapies such as mechanical circulatory support and cardiac transplantation should be considered in appropriate patients. Future research using AI driven algorithms for early diagnosis and treatment as well as development of newer genetic engineering technologies will drive improvements in diagnosis, treatment and patient outcomes.

Advances in HIV Gene Therapy.

Early gene therapy studies held great promise for the cure of heritable diseases, but the occurrence of various genotoxic events led to a pause in clinical trials and a more guarded approach to progress. Recent advances in genetic engineering technologies have reignited interest, leading to the approval of the first gene therapy product targeting genetic mutations in 2017. Gene therapy (GT) can be delivered either in vivo or ex vivo. An ex vivo approach to gene therapy is advantageous, as it allows for the characterization of the gene-modified cells and the selection of desired properties before patient administration. Autologous cells can also be used during this process which eliminates the possibility of immune rejection. This review highlights the various stages of ex vivo gene therapy, current research developments that have increased the efficiency and safety of this process, and a comprehensive summary of Human Immunodeficiency Virus (HIV) gene therapy studies, the majority of which have employed the ex vivo approach.

Nanomaterials for intelligent CRISPR-Cas tools: improving environment sustainability.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a desirable gene modification tool covering a wide area in various sectors of medicine, agriculture, and microbial biotechnology. The role of this incredible genetic engineering technology has been extensively investigated; however, it remains formidable with cargo choices, nonspecific delivery, and insertional mutagenesis. Various nanomaterials including lipid, polymeric, and inorganic are being used to deliver the CRISPR-Cas system. Progress in nanomaterials could potentially address these challenges by accelerating precision targeting, cost-effectiveness, and one-step delivery. In this review, we highlighted the advances in nanotechnology and nanomaterials as smart delivery systems for CRISPR-Cas so as to ameliorate applications for environmental remediation including biomedical research and healthcare, strategies for mitigating antimicrobial resistance, and to be used as nanofertilizers for enhancing crop growth, and reducing the environmental impact of traditional fertilizers. The timely co-evolution of nanotechnology and CRISPR technologies has contributed to smart novel nanostructure hybrids for improving the onerous tasks of environmental remediation and biological sustainability.

Genome-Wide Association Studies of Embryogenic Callus Induction Rate in Peanut (Arachis hypogaea L.).

The capability of embryogenic callus induction is a prerequisite for in vitro plant regeneration. However, embryogenic callus induction is strongly genotype-dependent, thus hindering the development of in vitro plant genetic engineering technology. In this study, to examine the genetic variation in embryogenic callus induction rate (CIR) in peanut (Arachis hypogaea L.) at the seventh, eighth, and ninth subcultures (T7, T8, and T9, respectively), we performed genome-wide association studies (GWAS) for CIR in a population of 353 peanut accessions. The coefficient of variation of CIR among the genotypes was high in the T7, T8, and T9 subcultures (33.06%, 34.18%, and 35.54%, respectively), and the average CIR ranged from 1.58 to 1.66. A total of 53 significant single-nucleotide polymorphisms (SNPs) were detected (based on the threshold value -log10(p) = 4.5). Among these SNPs, SNPB03-83801701 showed high phenotypic variance and neared a gene that encodes a peroxisomal ABC transporter 1. SNPA05-94095749, representing a nonsynonymous mutation, was located in the Arahy.MIX90M locus (encoding an auxin response factor 19 protein) at T8, which was associated with callus formation. These results provide guidance for future elucidation of the regulatory mechanism of embryogenic callus induction in peanut.

The Chinese Hamster Ovary Cell-Based H9 HA Subunit Avian Influenza Vaccine Provides Complete Protection against the H9N2 Virus Challenge in Chickens.

(1) Background: Avian influenza has attracted widespread attention because of its severe effect on the poultry industry and potential threat to human health. The H9N2 subtype of avian influenza viruses was the most prevalent in chickens, and there are several commercial vaccines available for the prevention of the H9N2 subtype of avian influenza viruses. However, due to the prompt antigenic drift and antigenic shift of influenza viruses, outbreaks of H9N2 viruses still continuously occur, so surveillance and vaccine updates for H9N2 subtype avian influenza viruses are particularly important. (2) Methods: In this study, we constructed a stable Chinese hamster ovary cell line (CHO) to express the H9 hemagglutinin (HA) protein of the major prevalent H9N2 strain A/chicken/Daye/DY0602/2017 with genetic engineering technology, and then a subunit H9 avian influenza vaccine was prepared using the purified HA protein with a water-in-oil adjuvant. (3) Results: The results showed that the HI antibodies significantly increased after vaccination with the H9 subunit vaccine in specific-pathogen-free (SPF) chickens with a dose-dependent potency of the immunized HA protein, and the 50 μg or more per dose HA protein could provide complete protection against the H9N2 virus challenge. (4) Conclusions: These results indicate that the CHO expression system could be a platform used to develop the subunit vaccine against H9 influenza viruses in chickens.

Microbial host engineering for sustainable isobutanol production from renewable resources.

Due to the limited resources and environmental problems associated with fossil fuels, there is a growing interest in utilizing renewable resources for the production of biofuels through microbial fermentation. Isobutanol is a promising biofuel that could potentially replace gasoline. However, its production efficiency is currently limited by the use of naturally isolated microorganisms. These naturally isolated microorganisms often encounter problems such as a limited range of substrates, low tolerance to solvents or inhibitors, feedback inhibition, and an imbalanced redox state. This makes it difficult to improve their production efficiency through traditional process optimization methods. Fortunately, recent advancements in genetic engineering technologies have made it possible to enhance microbial hosts for the increased production of isobutanol from renewable resources. This review provides a summary of the strategies and synthetic biology approaches that have been employed in the past few years to improve naturally isolated or non-natural microbial hosts for the enhanced production of isobutanol by utilizing different renewable resources. Furthermore, it also discusses the challenges that are faced by engineered microbial hosts and presents future perspectives to enhancing isobutanol production. KEY POINTS: • Promising potential of isobutanol to replace gasoline • Engineering of native and non-native microbial host for isobutanol production • Challenges and opportunities for enhanced isobutanol production.

THE LEGAL PERSPECTIVE ON CONSUMER PROTECTION OF GENETICALLY ENGINEERED FOOD PRODUCTS IN INDONESIA

The development of science and technology has triggered the use of genetic engineering. Genetic engineering is the transplantation of genes into other genes, both between genes and across genes, to produce valuable products for living creatures. Genetically engineered food products, due to the development of modern biotechnology in the field of genetic engineering technology, have given rise to new legal problems because of the possibility of posing a risk to human health. This research uses a normative juridical approach with an emphasis on secondary data. This research uses doctrinal research methods. The research specifications used are analytical descriptive. The research results show that there is no special legal protection for consumers of genetically engineered food products, because it still refers to consumer protection contained in the UUPK. It should be noted that genetic effects resulting from human genetic engineering need legal protection. Protection and intellectual property rights, where everyone, individually or collectively, must be a consumer of goods and services, so the parties need equal legal protection. Therefore, the responsibility of producers of genetically engineered food products for consumer losses must be able to fulfill the principle of justice because the economic position of producers is higher than that of consumers. By implementing the principle of absolute responsibility, the producer responsibility law will foster an attitude of caution for producers of genetically engineered food products to maintain the quality of their products.

Engineered Microglia‐Exosomes Coated Highly Twisting AIE Photothermal Agents to Efficiently Cross Blood‐Brain‐Barrier for Mild Photothermal‐Immune Checkpoint Blockade Therapy in Glioblastoma

AbstractImmune checkpoint blockade (ICB) therapy has achieved remarkable therapeutic effects in cancer, but it is not effective in glioblastoma (GBM). The main reason is that it is difficult for drugs to penetrate blood‐brain‐barrier (BBB) to GBM and lacks enough pre‐existing cytotoxic CD8+ T cells in GBM microenvironment. Here, two AIE photothermal agents (Fs and Fo) are synthesized with NIR‐II fluorescence emission and increase molecular twisting of AIE photothermal agents by simply changing one O atom into S atom, improving Fs photothermal conversion efficiency to 48%. Subsequently, engineered microglia‐exosomes AIE nanoparticles (EE@Fs‐NPs) are prepared by encapsulating Fs with microglia‐exosomes which express immune checkpoint LAG3 inhibitory antibody (anti‐LAG3) via genetic engineering technology. Engineered microglia‐exosomes endow that EE@Fs‐NPs cross BBB and target GBM, which successfully deliver anti‐LAG3 to GBM. Anti‐LAG3 highly reverses T cells exhaustion and generates TNF‐α for inhibiting the expression of heat shock proteins to enhance tumor cells thermosensitivity. Moreover, AIE photothermal agents of EE@Fs‐NPs generate mild photothermal therapy to destroy tumor cells and improve the infiltration of cytotoxic CD8+ T cells in GBM, which increases the responsiveness of ICB therapy. EE@Fs‐NPs produce powerful mild photothermal‐immune synergistic therapeutic effect, which will provide a therapeutic platform for the efficient treatment of GBM.

Elementary and Advanced Mechanisms for Genetic Engineering in Crops

Presently, the field of plant breeding is in the genomics era, where innovative techniques are being integrated to accelerate and enhance the efficiency of breeding. Traditional plant breeding methods rely on maintaining plant germplasm with desirable agronomic traits from distinct plants produced through crosses or mutagenesis. However, advancements in genetic engineering encompass all forms of genetic modification through recombinant DNA technology (RDT) and cell fusion mechanisms. These approaches shed light on areas involving mutant organisms, DNA replication, genetic linkage resolution, genetically modified organisms (GMOs), protein sequencing, functional genomics, and computational genomics alterations in genetic engineering. The integration of structural genomics into breeding and eugenics analysis has resulted in a vast knowledge base on crop genetics, species divergence, and molecular origin of traits, as well as the evolutionary history of crop lineage from ancient ancestral species. The genomic data and advancements have proven essential in identifying rare genes, alleles, or local lesions crucial to significant agronomic traits, thereby expediting breeding cycles. This article aims to explore the potential of emerging genetic engineering technologies, including synthetic biology and genome editing, to further advance crop genetics and plant breeding.

Enhancing Wheat Crop Yield and Quality through Genetic Engineering Technologies: A Comprehensive Review

The increasing world population has created an alarming situation to meet the demand for food. Ultimately wheat yield is required to be increased proportionally to the world’s population. An estimated increase of 05 tonnes per hectare is direly needed from the current production of 3.3 tonnes by the end of 2050. To achieve this goal it is required to adopt new, emerging, and efficient breeding techniques which caused a recent breakthrough in wheat genome sequencing and provided sufficient information for the traits which are directly related to quality and yield of crops. This review provides a comprehensive overview of tools and techniques which are widely used for discovering the functionality of genes that results in specific phenotypes of Wheat. It gives a deep historical account of the development of wheat transformation techniques and provides an idea about in which way these techniques have been adapted to produce gain-of-function phenotypes through gene overexpression, loss-of-function phenotypes through the expression of antisense RNAs (RNA interference or RNAi). Recently, gene structure and expression manipulation using site-specific nucleases such as CRISPR/Cas9 for genome editing. The review summarizes recent successes in the application of wheat genetic manipulation to enhance yield, improve wheat's nutritional and health-promoting qualities, and boost the crop's resistance to various biotic and abiotic stresses. Keywords: Transformation, gene knock-Out, gene knock-In, RNAI, overexpression.

A struggle for control beyond the facts: examining constructs of GM technology in Philippine opinion columns

ABSTRACT GM technology has constantly faced opposition in the Philippines. The debate heightened in 2016, when the Supreme Court halted the field testing of Bt eggplant, which triggered a public debate. This ruling was overturned, but GM technology remained in the public consciousness because of the general visibility of science, health, and food security issues. The researcher analyzed opinion columns in major Philippine news outlets from 2016-present to examine candid framings of the GM debate. Through inductive analysis, the researcher found that the debate represents a struggle for control. The crops are not so much monsters as they are hyperbolic jokes that do not meet their promises; they too, represent a foreign industrial invader that can be countered only by contextualized and localized farming. These findings add nuance to our understanding of the GM debate in the Global South.

Transformation of the p53 plasmid carrying Cas9 to Chili Callus (Capsicum annum) via Agrobacterium tumefaciens

Geminivirus is a virus that causes curly yellow disease in chili plants, which causes yield losses ranging from 20-100%. CRISPR/Cas9 facilitates the development of virus-resistant plants using genetic engineering technology. In this study, the process of transferring the plasmid p53/Cas9 to Agrobacterium was executed using the freeze-thawing technique. To validate the achievement of the transformation, an amplification process utilizing specific primers and an in-out primer combination was performed. We have achieved a transformation efficiency of 60%, as indicated by the PCR detection analysis of the putative recombinant callus. This outcome serves as a crucial starting point for the subsequent stages involved in transforming the p53 plasmid containing the Cas9 gene into chili (Capsicum annum) via Agrobacterium tumefaciens.

Status and developmental trends in recombinant collagen preparation technology.

Recombinant collagen is a pivotal topic in foundational biological research and epitomizes the application of critical bioengineering technologies. These technological advancements have profound implications across diverse areas such as regenerative medicine, organ replacement, tissue engineering, cosmetics and more. Thus, recombinant collagen and its preparation methodologies rooted in genetically engineered cells mark pivotal milestones in medical product research. This article provides a comprehensive overview of the current genetic engineering technologies and methods used in the production of recombinant collagen, as well as the conventional production process and quality control detection methods for this material. Furthermore, the discussion extends to foresee the strides in physical transfection and magnetic control sorting studies, envisioning an enhanced preparation of recombinant collagen-seeded cells to further fuel recombinant collagen production.