Characterization of CYP303A1 and its potential application based on ZIF-8 nanoparticle-wrapped dsRNA in Nilaparvata lugens (Stål).

BACKGROUND: RNA interference (RNAi) technology has been put forward as a promising method for pest control and resistance management. Mining highly efficient lethal genes and constructing stable double-stranded RNA (dsRNA) delivery systems are of great significance to improve the application potential of RNAi technology. RESULTS: In this study, we characterized a molting-related gene, NlCYP303A1, in Nilaparvata lugens that was highly expressed in the cuticle and at the end stages of each instar in nymphs. Silencing the expression of NlCYP303A1 in N. lugens resulted in a deformed phenotype and a significant increase in mortality. Furthermore, interfering with NlCYP303A1 changed the relative expression of key genes in the chitin synthesis and degradation pathway. Finally, we used the nanocarrier zeolitic imidazolate framework-8 (ZIF-8) to load dsNlCYP303A1, forming a complex denoted as dsNlCYP303A1@ZIF-8. The results of both feeding and rice-seedling dip experiments indicated that the expression of NlCYP303A1 was dramatically and persistently suppressed by the dsNlCYP303A1@ZIF-8 treatment, compared with treatment with dsNlCYP303A1, suggesting that ZIF-8 can enhance the interference efficiency as well as the stability of dsNlCYP303A1. CONCLUSIONS: Our results demonstrate that the lethal gene NlCYP303A1 can be employed as an excellent target for RNAi technology by loading onto a nano-delivery system, and provide new insights into the creation of innovative pest control approaches. © 2024 Society of Chemical Industry.

Collagen II enrichment through scAAV6-RNAi-mediated inhibition of matrix-metalloproteinases 3 and 13 in degenerative nucleus-pulposus cells degenerative disc disease and biological treatment strategies.

Intervertebral disc (IVD) degeneration damaging the extracellular matrix (ECM) of IVDs is the main cause of spine-associated disorders. Degenerative disc disease (DDD) is a multifaceted disorder, where environmental factors, inflammatory cytokines and catabolic enzymes act together. DDD starts typically due to imbalance between ECM biosynthesis and degradation within IVDs, especially through unbalanced degradation of aggrecan and collagen II in nucleus pulposus (NP). Current treatment approaches are primarily based on conservative or surgical therapies, which are insufficient for biological regeneration. The disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) and matrix metalloproteinases (MMPs) are the key proteolytic enzymes for degradation of aggrecan and collagens. Previously, high expression levels of ADAMTS4, ADAMTS5, MMP3 and MMP13, which are accompanied with low levels of aggrecan and collagen II, were demonstrated in degenerative human NP cells. Moreover, self-complementary adeno-associated virus type 6 (scAAV6) mediated inhibitions of ADAMTS4 and ADAMTS5 by RNA-interference (RNAi) could specifically enhance aggrecan level. Thus, MMPs are apparently the main degrading enzymes of collagen II in NP. Furthermore, scAAV6-mediated inhibitions of MMP3 and MMP13 have not yet been investigated. Therefore, we attempted to enhance the level of collagen II in degenerative NP cells by scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13. MRI was used to determine preoperative grading of IVD degeneration in patients. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting MMP3 or MMP13; and analysed by fluorescence microscopy, FACS, MTT assay, RT-qPCR, ELISA and western blotting. scAAV6-shRNRs have no impact on cell viability and proliferation, despite high transduction efficiencies (98.6%) and transduction units (1383 TU/Cell). Combined knockdown of MMP3 (92.8%) and MMP13 (90.9%) resulted in highest enhancement of collagen II (143.2%), whereby treatment effects were significant over 56 days (p < 0.001). Conclusively, scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13 help to progress less immunogenic and enduring biological treatments in DDD.

Immunological Roles of TmToll-2 in Response to Escherichia coli Systemic Infection in Tenebrio molitor.

The antimicrobial roles of Toll-like receptors have been mainly identified in mammalian models and Drosophila. However, its immunological function in other insects has yet to be fully clarified. Here, we determined the innate immune response involvement of TmToll-2 encountering Gram-negative, Gram-positive, and fungal infection. Our data revealed that TmToll-2 expression could be induced by Escherichia coli, Staphylococcus aureus, and Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of Tenebrio molitor young larvae. However, TmToll-2 silencing via RNAi technology revealed that sole E. coli systemic infection caused mortality in the double-strand RNA TmToll-2-injected group compared with that in the control group. Further investigation indicated that in the absence of TmToll-2, the final effector of Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated, mainly E. coli post-insult. We showed that the expression of all AMP genes was suppressed in the main immune organ of insects, namely, fat bodies, in silenced individuals, while the relevant expressions were not affected after fungal infection. Thus, our research revealed the immunological roles of TmToll-2 in different organs of T. molitor in response to pathogenic insults.

Nucleus-specific RNAi nanoplatform for targeted regulation of nuclear lncRNA function and effective cancer therapy.

In the context of cancer therapy, a recently identified therapeutic target is represented by the essential subtype of RNA transcripts - the long noncoding RNAs (lncRNA). While this is the case, it is especially difficult to successfully regulate the expression of this subtype in vivo, particularly due to the protection granted by the nuclear envelope of nuclear lncRNAs. This study documents the development of a nucleus-specific RNA interference (RNAi) nanoparticle (NP) platform for the targeted regulation of the nuclear lncRNA function, in order to effectuate successful cancer therapy. An NTPA (nucleus-targeting peptide amphiphile) and an endosomal pH-responsive polymer make up the novel RNAi nanoplatform in development, which is capable of complexing siRNA. The nanoplatform is capable of accumulating greatly in the tumor tissues and being internalized by tumor cells, following intravenous administration. The exposed complexes of the NTPA/siRNA may conveniently escape from the endosome with the pH-triggered NP disassociation, following which it can target the nucleus by specifically interacting with the importin α/ß heterodimer. In orthotopic and subcutaneous xenograft tumor models, this would result in a notable suppression of the expression of nuclear lncNEAT2 as well as greatly impede the growth of tumors in liver cancer.

Environmental biosafety assessment on transgenic Oncidium orchid modified by RNA interference of Phytoene Synthase genes.

Hybrid Oncidium orchids, such as Oncidium Gower Ramsey and Oncidium "Honey Angel," are popular cut flowers in Japan and Taiwan. Due to pollen sterility, no new varieties have been created by conventional breeding methods. Recently, we employed RNA interference (RNAi) technology to suppress phytoene synthase and successfully modified floret hue from yellow to white (Liu et al. 2019). Transgenic white Oncidium orchids, Honey Snow MF-1, have been grown to test their genetic stability, and their environmental biosafety was assessed for approximately one year under government regulatory instructions from the Council of Agriculture, Taiwan. In the present study, pollen sterility was demonstrated by cytological observation of the microsporogenesis step, pollen morphology abortion, and failure of pollen germination. Assays on allelopathic effect on the other plants and the soil rhizospheric microbial flora-revealed that transgenic Oncidium orchids are potentially safe with regard to environmental biodiversity. Therefore, the general release permissions have been granted and an application for licensing for commercial production is under way.

The Application of the RNA Interference Technologies for KRAS: Current Status, Future Perspective and Associated Challenges.

KRAS is a member of the murine sarcoma virus oncogene-RAS gene family. It plays an important role in the prevention, diagnosis and treatment of tumors during tumor cell growth and angiogenesis. KRAS is the most commonly mutated oncogene in human cancers, such as pancreatic cancers, colon cancers, and lung cancers. Detection of KRAS gene mutation is an important indicator for tracking the status of oncogenes, highlighting the developmental prognosis of various cancers, and the efficacy of radiotherapy and chemotherapy. However, the efficacy of different patients in clinical treatment is not the same. Since RNA interference (RNAi) technologies can specifically eliminate the expression of specific genes, these technologies have been widely used in the field of gene therapy for exploring gene function, infectious diseases and malignant tumors. RNAi refers to the phenomenon of highly specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. There are three classical RNAi technologies, including siRNA, shRNA and CRISPR-Cas9 system, and a novel synthetic lethal interaction that selectively targets KRAS mutant cancers. Therefore, the implementation of individualized targeted drug therapy has become the best choice for doctors and patients. Thus, this review focuses on the current status, future perspective and associated challenges in silencing of KRAS with RNAi technology.

Suppression of IGF1R in Melanoma Cells by an Adenovirus-Mediated One-Step Knockdown System.

Abnormal activation of the IGF1R signaling pathway accelerates melanoma development and metastases. RNAi systems with complex cloning procedures and unsatisfactory efficiency in suppressing gene expression have become the technical difficulties that hinder their utility when studying gene knockdown. Here we established a simplified adenovirus-mediated gene knockdown system by which a single adenoviral vector carries multiple siRNA fragments that can effectively suppress IGF1R expression in melanoma cells. We first generated the adenovirus that simultaneously expresses three human or mouse siRNAs targeting IGF1R (AdRIGF1R-OK). qRT-PCR and immunofluorescence staining revealed that IGF1R expression was significantly decreased in the melanoma cells that were infected with AdRIGF1R-OK. Bioluminescence imaging showed that the size of the tumor formed by the xenografts infected with AdRIGF1R-OK was significantly smaller than that of the controls. Annexin V-FITC flow cytometry assay, immunofluorescence staining for cleaved caspase-3, and Hoechst staining showed that more cells underwent apoptosis after infection with AdRIGF1R-OK. Luciferase reporter assay, crystal violet cell viability assay, and cell-cycle analysis showed that the proliferation of melanoma cells infected with AdRIGF1R-OK was significantly decreased compared to the controls. This study demonstrates that the OK system is effective in silencing gene expression, with promising potential to treat melanoma and other diseases.

New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli.

RNA interference-based technologies have emerged as an attractive and effective therapeutic option with potential application in diverse human diseases. These tools rely on the development of efficient strategies to obtain homogeneous non-coding RNA samples with adequate integrity and purity, thus avoiding non-targeted gene-silencing and related side-effects that impair their application onto pre-clinical practice. These RNAs have been preferentially obtained by in vitro transcription using DNA templates or via chemical synthesis. As an alternative to overcome the limitations presented by these methods, in vivo recombinant production of RNA biomolecules has become the focus in RNA synthesis research. Therefore, using pre-miR-29b as a model, here it is evaluated the time-course profile of Escherichia coli and Rhodovolum sulfidophilum microfactories to produce this microRNA. As the presence of major host contaminants arising from the biosynthesis process may have important implications in the subsequent downstream processing, it is also evaluated the production of genomic DNA and host total proteins. Considering the rapidly growing interest on these innovative biopharmaceuticals, novel, more cost-effective, simple and easily scaled-up technologies are highly desirable. As microRNA recombinant expression fulfills those requirements, it may take the leading edge in the methodologies currently available to obtain microRNAs for clinical or structural studies.

Recombinant baculovirus mediates dsRNA specific to rr2 delivery and its protective efficacy against WSSV infection.

White spot syndrome virus (WSSV) is a major causative agent in shrimp farming. Consequently, RNAi technology is an effective strategy to prevent WSSV infection in shrimp especially dsRNA targeting to rr2 of WSSV. In an effort to develop dsRNA expression in shrimp for control of WSSV infection, we developed a recombinant baculovirus expressing recombinant VP28 as the gene delivery system to carry a gene encoding dsRNA specific to rr2 for triggering the RNAi process in shrimp. The results showed that the recombinant baculovirus harboring VP28 was able to express VP28 indicated by Western blot with polyclonal antibody specific to VP28. VP28 transcript was detected in shrimp hemocytes after co-culture hemocytes with the recombinant baculovirus displaying VP28. In addition, we found that shrimp injected with the recombinant baculovirus displaying VP28 and encoding dsRNA synthetic gene specific to rr2 (Bac-VP28-dsrr2) showed the lowest cumulative mortality (33%) at 14days post infection (dpi) when compared to shrimp injected with baculovirus displaying VP28 (Bac-VP28) (64% cumulative mortality) (p<0.05). According to the results, shrimp injected with Bac-VP28-dsrr2 also showed significantly lower WSSV copies than shrimp injected with Bac-VP28 (p<0.05) along with the down-regulation of rr2 expression at 1, 3 and 7dpi. In conclusion, the Bac-VP28-dsrr2 was effective in prevention of WSSV infection. Therefore, the results obtained here can be applied to the prevention of WSSV infection by mixing the recombinant baculovirus with shrimp feed in the future.

Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilum.

The present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025 µg of plasmid DNA, with a heat-shock of 2 min at 35 °C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182 µg/L, after 40 h of bacterial growth, and the total intracellular pre-miR-29b was of about 358 µg/L, at 32 h. At the end of the fermentation, it was verified that almost 87 % of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics.

The potential of class 3 semaphorins as both targets and therapeutics in cancer.

INTRODUCTION: Semaphorins have been originally identified as a family of evolutionary conserved soluble or membrane-associated proteins involved in diverse developmental phenomena. This family of proteins profoundly influences numerous pathophysiological processes, including organogenesis, cardiovascular development and immune response. Apart from steering the neural networking process, these are implicated in a broad range of biological operations including regulation of tumor progression and angiogenesis. AREAS COVERED: Members of class 3 semaphorin family are known to modulate various cellular processes involved in malignant transformation. Some of the family members trigger diverse signaling processes involved in tumor progression and angiogenesis by binding with plexin and neuropilin. A better understanding of the various signaling mechanisms by which semaphorins modulate tumor progression and angiogenesis may serve as crucial tool in crafting new semaphorin-based anticancer therapy. These include treatment with recombinant tumor suppressive semaphorins or inhibition of tumor-promoting semaphorins by their specific siRNAs, small-molecule inhibitors or specific receptors using neutralizing antibodies or blocking peptides that might serve as novel strategies for effective management of cancers. EXPERT OPINION: This review focuses on all the possible avenues to explore various members of class 3 semaphorin family to serve as therapeutics for combating cancer.

Advances in Anti-Cancer Drug Development Targeting Carbonic Anhydrase IX and XII.

The microenvironment within a solid tumor is heterogeneous with regions being both acidic and hypoxic. As a result of this, cancer cells upregulate genes that allow survival in such environments. Some of these genes are pH regulatory factors, including carbonic anhydrase IX (CA IX) and in some cases XII (CA XII). CA IX helps to maintain normal cytoplasmic pH (pHi) while simultaneously contributing to the extracellular pH (pHe). CA XII is also thought to be responsible for stabilizing pHe at physiological conditions. Extracellular acidification of the tumor microenvironment promotes local invasion and metastasis while decreasing the effectiveness of adjuvant therapies, thus contributing to poor cancer clinical outcomes. In this review, we describe the properties of CA IX and CA XII that substantiate their potential use as anticancer targets. We also discuss the current status of CA isoform-selective inhibitor development and patents of CA IX/XII targeted inhibitors that show potential for treating aggressive tumors. Some of the recently published patents discussed include sulfonamide-based small molecule inhibitors including derivatives of boron cluster compounds; metal complexes of poly(carboxyl)amine-containing ligands; nitroi-midazole-, ureidosulfonamide-, and coumarin-based compounds; as well as G250 and A610 monoclonal antibodies for cancer treatment.

Osteopontin as a therapeutic target for cancer.

INTRODUCTION: Cancer is a complex pathological disorder, established as a result of accumulation of genetic and epigenetic changes, which lead to adverse alterations in the cellular phenotype. Tumor progression involves intricate signaling mediated through crosstalk between various growth factors, cytokines and chemokines. Osteopontin (OPN), a chemokine-like protein, is involved in promotion of neoplastic cancer into higher grade malignancies by regulating various facets of tumor progression such as cell proliferation, angiogenesis and metastasis. AREAS COVERED: Tumors as well as stroma-derived OPN play key roles in various signaling pathways involved in tumor growth, angiogenesis and metastasis. OPN derived from tumor-activated macrophages modulates the tumor microenvironment and thereby regulate melanoma growth and angiogenesis. OPN also regulates hypoxia-inducible factor-1α-dependent VEGF expression leading to breast tumor growth and angiogenesis in response to hypoxia. Thus, a clear understanding of the molecular mechanism underlying OPN-mediated regulation will shed light on exciting avenues for further investigation of targeted therapies. Silencing of OPN using RNAi technology, blocking OPN activity using specific antibodies and small-molecule inhibitors might provide novel strategies, which would aid in developing effective therapeutics for the treatment of various types of cancer. EXPERT OPINION: This review focuses on new possibilities to exploit OPN as a tumor and stroma-derived therapeutic target to combat cancer.