Engineered Mesenchymal Stem Cells as Treatment for Cancers: Opportunities, Clinical Applications and Challenges.

The insufficient and unspecific target of classical chemotherapies often leads to therapy resistance and cancer recurrence. Over the past decades, discoveries about mesenchymal stem cell (MSC) biology have provided new potential approaches to improve cancer therapy. Researchers have utilised the multipotent, regenerative and immunosuppressive qualities of MSCs and tropisms towards inflammatory, hypoxic and malignant sites in various therapeutic applications. Although MSC-based therapies have generally been demonstrated safe, their effectiveness remains limited when these cells are used alone. However, through genetic engineering, researchers have proven that MSCs can be modified to have specialised delivery roles to increase their therapeutic efficacy in cancer treatment. They can be made to overexpress therapeutic proteins through viral or non-viral genetic modification, which enhances their innate properties. Nevertheless, these engineering strategies must be optimised to increase therapeutic efficacy and targeting effectiveness while minimising any loss of MSC function. This review underscores the cutting-edge methods for engineering MSCs, discusses their promise and the difficulties in translating them into clinical settings, and offers some prospective suggestions for the future on achieving their full therapeutic potential.

Genetically modified mesenchymal stromal cells: a cell-based therapy offering more efficient repair after myocardial infarction.

Myocardial infarction (MI) is a serious complication of coronary artery disease. This condition is common worldwide and has a profound impact on patients' lives and quality of life. Despite significant advances in the treatment of heart disease in modern medicine, the efficient treatment of MI still faces a number of challenges. Problems such as scar formation and loss of myocardial function after a heart attack still limit patients' recovery. Therefore, the search for a new therapeutic tool that can promote repair and regeneration of myocardial tissue has become crucial. In this context, mesenchymal stromal cells (MSCs) have attracted much attention as a potential therapeutic tool. MSCs are a class of adult stem cells with multidirectional differentiation potential, derived from bone marrow, fat, placenta and other tissues, and possessing properties such as self-renewal and immunomodulation. The application of MSCs may provide a new direction for the treatment of MI. These stem cells have the potential to differentiate into cardiomyocytes and vascular endothelial cells in damaged tissue and to repair and protect myocardial tissue through anti-inflammatory, anti-fibrotic and pro-neovascularization mechanisms. However, the clinical results of MSCs transplantation for the treatment of MI are less satisfactory due to the limitations of the native function of MSCs. Genetic modification has overcome problems such as the low survival rate of transplanted MSCs in vivo and enhanced their functions of promoting neovascularization and differentiation into cardiomyocytes, paving the way for them to become an effective tool for repair therapy after MI. In previous studies, MSCs have shown some therapeutic potential in experimental animals and preliminary clinical trials. This review aims to provide readers with a comprehensive and in-depth understanding to promote the wider application of engineering MSCs in the field of MI therapy, offering new hope for recovery and improved survival of cardiac patients.

Mical1 deletion in tyrosinase expressing cells affects mouse running gaits.

Neuronal development is a highly regulated process that is dependent on the correct coordination of cellular responses to extracellular cues. In response to semaphorin axon guidance proteins, the MICAL1 protein is stimulated to produce reactive oxygen species that oxidize actin on specific methionine residues, leading to filamentous actin depolymerization and consequent changes in neuronal growth cone dynamics. Crossing genetically modified mice homozygous for floxed Mical1 (Mical1fl/fl) alleles with transgenic mice expressing Cre recombinase under the control of a tyrosinase gene enhancer/promoter (Tyr::Cre) enabled conditional Mical1 deletion. Immunohistochemical analysis showed Mical1 expression in the cerebellum, which plays a prominent role in the coordination of motor movements, with reduced Mical1 expression in Mical1fl/fl mice co-expressing Tyr::Cre. Analysis of the gaits of mice running on a treadmill showed that both male and female Mical1fl/fl, Tyr::Cre mutant mice had significant alterations to their striding patterns relative to wild-type mice, although the specific aspects of their altered gaits differed between the sexes. Additional motor tests that involved movement on a rotating rod, descending a vertical pole, or crossing a balance beam did not show significant differences between the genotypes, suggesting that the effect of the Mical1fl/fl, Tyr::Cre genetic modifications was only manifested during specific highly coordinated movements that contribute to running. These findings indicate that there is a behavioral consequence in Mical1fl/fl, Tyr::Cre mutant mice that affects motor control as manifested by alterations in their gait.

The molecular mechanism of NF-κB dysregulation across different subtypes of renal cell carcinoma.

BACKGROUND: The nuclear factor kappa B (NF-κB) is a critical pathway that regulates various cellular functions, including immune response, proliferation, growth, and apoptosis. Furthermore, this pathway is tightly regulated to ensure stability in the presence of immunogenic triggers or genotoxic stimuli. The lack of control of the NF-κB pathway can lead to the initiation of different diseases, mainly autoimmune diseases and cancer, including Renal cell carcinoma (RCC). RCC is the most common type of kidney cancer and is characterized by complex genetic composition and elusive molecular mechanisms. AIM OF REVIEW: The current review summarizes the mechanism of NF-κB dysregulation in different subtypes of RCC and its impact on pathogenesis. KEY SCIENTIFIC CONCEPT OF REVIEW: This review highlights the prominent role of NF-κB in RCC development and progression by driving the expression of multiple genes and interplaying with different pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In silico analysis of RCC cohorts and molecular studies have revealed that multiple NF-κB members and target genes are dysregulated. The dysregulation includes receptors such as TLR2, signal-transmitting members including RelA, and target genes, for instance, HIF-1α. The lack of effective regulatory mechanisms results in a constitutively active NF-κB pathway, which promotes cancer growth, migration, and survival. In this review, we comprehensively summarize the role of dysregulated NF-κB-related genes in the most common subtypes of RCC, including clear cell RCC (ccRCC), chromophobe RCC (chRCC), and papillary RCC (PRCC).

Methodological Approaches for Increasing the Retroviral Transduction Efficiency of Primary NK Cells.

BACKGROUND: The growing attention to NK cells for cancer cell therapy is associated with the need to establish highly efficient protocols for their genetic modification, particularly by retroviral transduction. OBJECTIVE: In this work, we have optimized several stages of the retroviral-based modification process, and determined the distribution of the amino acid transporter ASCT2 between NK cell subsets. METHODS: Retroviral particles were produced using the Phoenix Ampho cell line transfected with the calcium phosphate method . We used RD114-based retroviral transduction for lymphocyte cell lines and primary NK cells. RESULTS: We have determined the optimal time to collect the RD114-pseudotyped viral supernatants resulting in the titer of viral particles required for efficient NK cell modification to be between 48 and 72 hours. Retroviral modification by retronectin-based method did not alter NK cell functional activity and cell survival. We identified differences in the Multiplicity of Infection (MOI) among cell lines that were partially associated with the ASCT2 surface expression. Cells with higher ASCT2 levels were more susceptible to transduction with RD114-pseudotyped viral particles. Higher ASCT2 expression levels were revealed in activated CD57+ and KIR2DL2DL3+ NK cells compared to their negative counterparts. CONCLUSION: Our findings provide a more nuanced understanding of NK cell transduction, offering valuable insights for improving therapeutic applications involving NK cell modification.

Enhancing endothelial colony-forming cells for treating diabetic vascular complications: challenges and clinical prospects.

Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia, leading to various vascular complications. Accumulating evidence indicates that endothelial colony-forming cells (ECFCs) have attractive prospects for repairing and restoring blood vessels. Thus, ECFCs may be a novel therapeutic option for diabetic patients with vascular complications who require revascularization therapy. However, it has been reported that the function of ECFCs is impaired in DM, which poses challenges for the autologous transplantation of ECFCs. In this review, we summarize the molecular mechanisms that may be responsible for ECFC dysfunction and discuss potential strategies for improving the therapeutic efficacy of ECFCs derived from patients with DM. Finally, we discuss barriers to the use of ECFCs in human studies in light of the fact that there are no published reports using these cells in humans.

Co-mutation of OsLPR1/3/4/5 provides a promising strategy to minimize Cd contamination in rice grains.

Minimizing cadmium (Cd) contamination in rice grains is crucial for ensuring food security and promoting sustainable agriculture. Utilizing genetic modification to generate rice varieties with low Cd accumulation is a promising strategy due to its cost-effectiveness and operational simplicity. Our study demonstrated that the CRISPR-Cas9-mediated quadruple mutation of the multicopper oxidase genes OsLPR1/3/4/5 in the japonica rice cultivar Tongjing 981 had little effect on yields. However, a notable increase was observed in the cell wall functional groups that bind with Cd. As a result, the quadruple mutation of OsLPR1/3/4/5 enhanced Cd sequestration within the cell wall while reducing Cd concentrations in both xylem and phloem sap, thereby inhibiting Cd transport from roots to shoots. Consequently, Cd concentrations in brown rice and husk in oslpr1/3/4/5 quadruple mutants (qm) decreased by 52% and 55%, respectively, compared to the wild-type. These findings illustrate that the quadruple mutation of OsLPR1/3/4/5 is an effective method for minimizing Cd contamination in rice grains without compromising yields. Therefore, the quadruple mutation of OsLPR1/3/4/5 via biotechnological pathways may represent a valuable strategy for the generation of new rice varieties with low Cd accumulation.

Engineering a One Health Super Wheat.

Wheat is the predominant crop worldwide, contributing approximately 20% of protein and calories to the human diet. However, the yield potential of wheat faces limitations due to pests, diseases, and abiotic stresses. Although conventional breeding has improved desirable traits, the use of modern transgenesis technologies has been limited in wheat in comparison to other crops such as maize and soybean. Recent advances in wheat gene cloning and transformation technology now enable the development of a super wheat consistent with the One Health goals of sustainability, food security, and environmental stewardship. This variety combines traits to enhance pest and disease resistance, elevate grain nutritional value, and improve resilience to climate change. In this review, we explore ways to leverage current technologies to combine and transform useful traits into wheat. We also address the requirements of breeders and legal considerations such as patents and regulatory issues.

Heart immunoengineering by lentiviral vector-mediated genetic modification during normothermic ex vivo perfusion.

Heart transplantation is associated with major hurdles, including the limited number of available organs for transplantation, the risk of rejection due to genetic discrepancies, and the burden of immunosuppression. In this study, we demonstrated the feasibility of permanent genetic engineering of the heart during ex vivo perfusion. Lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin (shß2m) and class II transactivator (shCIITA) were delivered to the graft during two hours of normothermic EVHP. Highly efficient genetic engineering was indicated by stable reporter gene expression in endothelial cells and cardiomyocytes. Remarkably, swine leucocyte antigen (SLA) class I and SLA class II expression levels were decreased by 66% and 76%, respectively, in the vascular endothelium. Evaluation of lactate, troponin T, and LDH levels in the perfusate and histological analysis showed no additional cell injury or tissue damage caused by lentiviral vectors. Moreover, cytokine secretion profiles (IL-6, IL-8, and TNF-α) of non-transduced and lentiviral vector-transduced hearts were comparable. This study demonstrated the ex vivo generation of genetically engineered hearts without compromising tissue integrity. Downregulation of SLA expression may contribute to reduce the immunogenicity of the heart and support graft survival after allogeneic or xenogeneic transplantation.

Microbial Biofilms: Features of Formation and Potential for Use in Bioelectrochemical Devices.

Microbial biofilms present one of the most widespread forms of life on Earth. The formation of microbial communities on various surfaces presents a major challenge in a variety of fields, including medicine, the food industry, shipping, etc. At the same time, this process can also be used for the benefit of humans-in bioremediation, wastewater treatment, and various biotechnological processes. The main direction of using electroactive microbial biofilms is their incorporation into the composition of biosensor and biofuel cells This review examines the fundamental knowledge acquired about the structure and formation of biofilms, the properties they have when used in bioelectrochemical devices, and the characteristics of the formation of these structures on different surfaces. Special attention is given to the potential of applying the latest advances in genetic engineering in order to improve the performance of microbial biofilm-based devices and to regulate the processes that take place within them. Finally, we highlight possible ways of dealing with the drawbacks of using biofilms in the creation of highly efficient biosensors and biofuel cells.

The bioproduction of astaxanthin: A comprehensive review on the microbial synthesis and downstream extraction.

Astaxanthin is a valuable orange-red carotenoid with wide applications in agriculture, food, cosmetics, pharmaceuticals and nutraceuticals areas. At present, the biological synthesis of astaxanthin mainly relies on Haematococcus pluvialis and Xanthophyllomyces dendrorhous. With the rapid development of synthetic biology, more recombinant microbial hosts have been genetically constructed for astaxanthin production including Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica. As multiple genes (15) were involved in the astaxanthin synthesis, it is particularly important to adopt different strategies to balance the metabolic flow towards the astaxanthin synthesis. Furthermore, astaxanthin is a fat-soluble compound stored intracellularly, hence efficient extraction methods are also essential for the economical production of astaxanthin. Several efficient and green extraction methods of astaxanthin have been reported in recent years, including the superfluid extraction, ionic liquid extraction and microwave-assisted extraction. Accordingly, this review will comprehensively introduce the advances on the astaxanthin production and extraction by using different microbial hosts and strategies to improve the astaxanthin synthesis and extraction efficiency.

Possibilities and efficiency of MSC co-transfection for gene therapy.

Mesenchymal stem/stromal cells (MSCs) are not only capable of self-renewal, trans-differentiation, homing to damaged tissue sites and immunomodulation by secretion of trophic factors but are also easy to isolate and expand. Because of these characteristics, they are used in numerous clinical trials for cell therapy including immune and neurological disorders, diabetes, bone and cartilage diseases and myocardial infarction. However, not all trials have successful outcomes, due to unfavourable microenvironmental factors and the heterogenous nature of MSCs. Therefore, genetic manipulation of MSCs can increase their prospect. Currently, most studies focus on single transfection with one gene. Even though the introduction of more than one gene increases the complexity, it also increases the effectivity as different mechanism are triggered, leading to a synergistic effect. In this review we focus on the methodology and efficiency of co-transfection, as well as the opportunities and pitfalls of these genetically engineered cells for therapy.

Molecular Mechanisms of CBL-CIPK Signaling Pathway in Plant Abiotic Stress Tolerance and Hormone Crosstalk.

Abiotic stressors, including drought, salt, cold, and heat, profoundly impact plant growth and development, forcing elaborate cellular responses for adaptation and resilience. Among the crucial orchestrators of these responses is the CBL-CIPK pathway, comprising calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs). While CIPKs act as serine/threonine protein kinases, transmitting calcium signals, CBLs function as calcium sensors, influencing the plant's response to abiotic stress. This review explores the intricate interactions between the CBL-CIPK pathway and plant hormones such as ABA, auxin, ethylene, and jasmonic acid (JA). It highlights their role in fine-tuning stress responses for optimal survival and acclimatization. Building on previous studies that demonstrated the enhanced stress tolerance achieved by upregulating CBL and CIPK genes, we explore the regulatory mechanisms involving post-translational modifications and protein-protein interactions. Despite significant contributions from prior research, gaps persist in understanding the nuanced interplay between the CBL-CIPK system and plant hormone signaling under diverse abiotic stress conditions. In contrast to broader perspectives, our review focuses on the interaction of the pathway with crucial plant hormones and its implications for genetic engineering interventions to enhance crop stress resilience. This specialized perspective aims to contribute novel insights to advance our understanding of the potential of the CBL-CIPK pathway to mitigate crops' abiotic stress.

Application of Oncolytic Poxviruses: An Emerging Paradigm in Cancer Therapy.

Despite the significant advancement of new tools and technology in the field of medical biology and molecular biology, the challenges in the treatment of most cancer types remain constant with the problem of developing resistance toward drugs and no substantial enhancement in the overall survival rate of cancer patients. Immunotherapy has shown the most promising results in different clinical and preclinical trials in the treatment of various cancer due to its higher efficacy and minimum collateral damage in many cancer patients as compared to conventional chemotherapy and radiotherapy. An oncolytic virus is a new class of immunotherapy that can selectively replicate in tumor cells and destroy them by the process of cell lysis while exerting minimum or no effect on a normal cell. Besides this, it can also activate the host's innate immune system, which generates an anti-tumor immune response to eliminate the tumor cells. Several wild types and genetically modified viruses have been investigated to show oncolytic behavior. Vaccinia virus has been studied extensively and tested for its promising oncolytic nature on various model systems and clinical trials. Recently, several engineered vaccinia viruses have been developed that express the desired genes encoded for selective penetration in tumor cells and enhanced activation of the immune system for generating anti-tumor immunity. However, further investigation is required to prove their potential and enhance their therapeutic efficacy.

Genetic Transformation of Cryptococcus Species with Agrobacterium Transfer DNA.

Transformation of foreign DNA into Cryptococcus species is a powerful tool for exploring gene functions in these human pathogens. Agrobacterium tumefaciens-mediated transformation (AtMT) has been used for the stable introduction of exogenous DNA into Cryptococcus for over two decades, being particularly impactful for insertional mutagenesis screens to discover new genes involved in fungal biology. A detailed protocol to conduct this transformation method is provided in the chapter. Scope for modifications and the benefits and disadvantages of using AtMT in Cryptococcus species are also presented.

Challenges for the Post-Market Environmental Monitoring in the European Union Imposed by Novel Applications of Genetically Modified and Genome-Edited Organisms.

Information on the state of the environment is important to achieve the objectives of the European Green Deal, including the EU's Biodiversity Strategy for 2030. The existing regulatory provisions for genetically modified organisms (GMOs) foresee an obligatory post-market environmental monitoring (PMEM) of potential adverse effects upon release into the environment. So far, GMO monitoring activities have focused on genetically modified crops. With the advent of new genomic techniques (NGT), novel GMO applications are being developed and may be released into a range of different, non-agricultural environments with potential implications for ecosystems and biodiversity. This challenges the current monitoring concepts and requires adaptation of existing monitoring programs to meet monitoring requirements. While the incorporation of existing biodiversity monitoring programs into GMO monitoring at the national level is important, additional monitoring activities will also be required. Using case examples, we highlight that monitoring requirements for novel GMO applications differ from those of GM crop plants previously authorized for commercial use in the European Union.

Global status of gene edited animals for agricultural applications.

Gene editing (GnEd) involves using a site-directed nuclease to introduce a double-strand break (DSB) at a targeted location in the genome. A literature search was performed on the use of GnEd in animals for agricultural applications. Data was extracted from 212 peer-reviewed articles that described the production of at least one living animal employing GnEd technologies for agricultural purposes. The most common GnEd system reported was CRISPR/Cas9, and the most frequent type of edit was the unguided insertion or deletion resulting from the repair of the targeted DSB leading to a knock-out (KO) mutation. Animal groups included in the reviewed papers were ruminants (cattle, sheep, goats, n=63); monogastrics (pigs and rabbits, n=60); avian (chicken, duck, quail, n=17); aquatic (many species, n=65), and insects (honeybee, silkworm, n=7). Yield (32%), followed by reproduction (21%) and disease resistance (17%) were the most commonly targeted traits. Over half of the reviewed papers had Chinese first-authorship. Several countries, including Argentina, Australia, Brazil, Colombia and Japan, have adopted a regulatory policy that considers KO mutations introduced following GnEd DSB repair as akin to natural genetic variation, and therefore treat these GnEd animals analogously to those produced using conventional breeding. This approach has resulted in a non-GMO determination for a small number of GnEd food animal applications, including three species of GnEd KO fast-growing fish, (red sea bream, olive flounder and tiger pufferfish in Japan), KO fish and cattle in Argentina and Brazil, and porcine reproductive and respiratory syndrome (PRRS) virus disease-resistant KO pigs in Colombia.

Transposon insertion in Rothia dentocariosa.

OBJECTIVES: Rothia spp. are emerging as significant bacteria associated with oral health, with Rothia dentocariosa being one of the most prevalent species. However, there is a lack of studies examining these properties at the genetic level. This study aimed to establish a genetic modification platform for R. dentocariosa. METHODS: Rothia spp. were isolated from saliva samples collected from healthy volunteers. Subsequently, R. dentocariosa strains were identified through colony morphology, species-specific polymerase chain reaction (PCR), and 16S ribosomal RNA gene sequencing. The identified strains were then transformed with plasmid pJRD215, and the most efficient strain was selected. Transposon insertion mutagenesis was performed to investigate the possibility of genetic modifications. RESULTS: A strain demonstrating high transforming ability, designated as R. dentocariosa LX16, was identified. This strain underwent transposon insertion mutagenesis and was screened for 5-fluoroorotic acid-resistant transposants. The insertion sites were confirmed using arbitrary primed PCR, gene-specific PCR, and Sanger sequencing. CONCLUSION: This study marks the first successful genetic modification of R. dentocariosa. Investigating R. dentocariosa at the genetic level can provide insights into its role within the oral microbiome.