Transfection Of Cells Research Articles

Enhancing wound healing and overcoming cisplatin resistance in ovarian cancer.

Ovarian cancer (OC) poses significant oncological challenges, notably impaired wound healing in the context of cisplatin (DDP) resistance. This study investigates the role of miR-200b in OC, emphasizing its impact on wound healing processes through DNMT3A/TGF-β1 pathway. The primary aim was to explore how miR-200b regulates autophagy and its consequential effects on wound healing in OC, alongside its influence on cisplatin resistance. Utilizing DDP-sensitive (A2780) and resistant (A2780/DDP) OC cell lines, along with human fibroblast cultures, the study employed an array of in vitro techniques. These included cell transfection with miR-200b mimic or inhibitor, chromatin immunoprecipitation (ChIP), dual-luciferase reporter (DLR) assays, quantitative PCR, Western blotting, MTT and particularly, wound healing assays. The research highlighted the role of miR-200b in wound healing within OC. Inhibition of miR-200b in A2780 cells and its mimic in A2780/DDP cells affected cell viability, indicating the link with DDP resistance. Crucially, miR-200b mimic significantly delayed fibroblast-mediated wound closure in assays, underscoring its impact on wound healing. Bioinformatics analysis and subsequent DLR assays confirmed miR-200b's interaction with DNMT3A, affecting TGF-β1 expression, the key factor in wound repair. Further, ChIP, quantitative PCR and Western blot analyses validated the interaction and expression changes in DNMT3A and TGF-β1. The study demonstrated that miR-200b played a pivotal role in OC by modulating autophagy, which in turn significantly affected wound healing through the DNMT3A/TGF-β1 pathway.

Variable antiviral activity of islatravir against M184I/V mutant HIV-1 selected during antiretroviral therapy.

Islatravir is a new antiretroviral drug that inhibits the reverse transcriptase (RT) of HIV-1 through multiple mechanisms. It is proposed to be used in combination with doravirine, a new NNRTI. M184V/I mutations have been shown to reduce the in vitro antiviral activity of islatravir, but their effect when pre-selected during ART has not been investigated. HIV-1 rt sequences were obtained from four individuals of the Garrahan HIV cohort prior to, or during virological failure to ART. HIV-1 infectious molecular clones were constructed on an NL4-3 backbone, and infectious viruses were produced by transfection of 293T cells. Fold-changes in IC50 were calculated for each mutant versus the NL4-3 WT. HIV-1 phenotypic drug resistance was tested in vitro against NRTIs and NNRTIs. In all the cases, M184I/V, either alone or in the presence of other mutations, was associated with reduced susceptibility to islatravir, abacavir and lamivudine. Viruses carrying M184V/I showed variable levels of resistance to islatravir (4.8 to 33.8-fold). The greatest reduction in susceptibility was observed for viruses carrying the mutations M184V + V106I (33.8-fold resistance) or M184V + I142V (25.2-fold resistance). For NNRTIs, the presence of V106I alone did not affect susceptibility to doravirine or etravirine, but showed a modest reduction in susceptibility to efavirenz (6-fold). Susceptibility to doravirine was slightly reduced only for one of the mutants carrying V106I in combination with Y181C and M184V. Mutations and polymorphisms selected in vivo together with M184V/I depend on the viral genetic context and on ART history, and could affect the efficacy of islatravir once available for use in the clinic.

Understanding Pseudophosphatase Function Through Biochemical Interactions.

Pseudophosphatases have been solidified as important signaling molecules that regulate signal transduction cascades. However, their mechanisms of action remain enigmatic. Reflecting this mystery, the prototypical pseudophosphatase STYX (phospho-serine-threonine/tyrosine-binding protein) was named with allusion to the river of the dead in Greek mythology to emphasize that these molecules are "dead" phosphatases. Although proteins with STYX domains do not catalyze dephosphorylation, this does not preclude their having other functions, including as integral elements of signaling networks. Thus, understanding their roles may mark them as potential novel drug targets. This chapter outlines common strategies used to characterize the functions of pseudophosphatases, using as an example MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine-threonine/tyrosine-binding], which has been linked to tumorigenesis, hepatocellular carcinoma, glioblastoma, apoptosis, and neuronal differentiation. We start with the importance of "restoring" (when possible) phosphatase activity in a pseudophosphatase, so the active mutant may be used as a comparison control throughout immunoprecipitation and mass spectrometry analyses. To this end, we provide protocols for site-directed mutagenesis, mammalian cell transfection, co-immunoprecipitation, phosphatase activity assays, and immunoblotting that we have used to investigate MK-STYX and the active mutant MK-STYXactive. We also highlight the importance of utilizing RNA interference (RNAi) "knockdown" technology to determine a cellular phenotype in various cell lines. Therefore, we outline our protocols for introducing short hairpin RNA (shRNA) expression plasmids into mammalian cells and quantifying knockdown of gene expression with real-time quantitative PCR (qPCR). We also provide a bioinformatic approach to investigating MK-STYX and MK-STYX(active mutant). These bioinformatic approaches can stand alone experimentally but also complement and enhance "wet" bench approaches such as binding assays and/or activity assays. A combination of cellular, molecular, biochemical, proteomic, and bioinformatic techniques has been a powerful tool in identifying novel functions of MK-STYX. Likewise, the information provided here should be a helpful guide to elucidating the functions of other pseudophosphatases.

LncRNA-mir3471-limd1 regulatory network plays critical roles in HIBD.

The purpose of this study was to identify the target genes of tcon_00044595, elucidate its activation site, and provide novel insights into the pathogenesis and treatment of neonatal hypoxic-ischemic brain damage (HIBD). Through homologous blast analysis, we identified predicted target sequences in the neighboring regions of the long non-coding RNA (lncRNA) tcon_00044595, suggesting that limd1 is its target gene. Starbase was utilized to identify potential candidate microRNAs associated with the lncRNA. The interaction between the candidate microRNAs and limd1 was investigated and validated using various experimental methods including in vitro cell culture, cell transfection, dual fluorescence reporter detection system, and real-time PCR. Homology alignment analysis revealed that the lncRNA tcon_00044595 exhibited a 246bp homologous sequence at the 3' end of the adjacent limd1 gene, with a conservation rate of 68%. Analysis conducted on Starbase online identified three potential microRNA candidates: miR-3471, miR-883a-5p, and miR-214-3p. Intracellular expression of the limd1 gene was significantly down-regulated upon transfection with miR-3471, while the other two microRNAs did not produce noticeable effects. Luciferase reporter assays identified two interaction sites (UTR-1, UTR-2) between miR-3471 and the limd1 3'UTR, with UTR-1 exhibiting a strong influence. Further CCK8 assay indicated a protective role of miR-3471 during low oxygen stroke in HIBD. The potential regulatory relationship between lncRNA (tcon_00044595), miR-3471, and the target gene limd1 suggests their involvement in the occurrence and development of HIBD, providing new insights for investigating the underlying mechanisms and exploring targeted therapeutic approaches for HIBD.

The impact of S2 mutations on Omicron SARS-CoV-2 cell surface expression and fusogenicity

ABSTRACT SARS-CoV-2 Omicron subvariants are still emerging and spreading worldwide. These variants contain a high number of polymorphisms in the spike (S) glycoprotein that could potentially impact their pathogenicity and transmission. We have previously shown that the S:655Y and P681H mutations enhance S protein cleavage and syncytia formation. Interestingly, these polymorphisms are present in Omicron S protein. Here, we characterized the cleavage efficiency and fusogenicity of the S protein of different Omicron sublineages. Our results showed that Omicron BA.1 subvariant is efficiently cleaved but it is poorly fusogenic compared to previous SARS-CoV-2 strains. To understand the basis of this phenotype, we generated chimeric S protein using combinations of the S1 and S2 domains from WA1, Delta and Omicron BA.1 variants. We found that the S2 domain of Omicron BA.1 hindered efficient cell–cell fusion. Interestingly, this domain only contains six unique polymorphisms never detected before in ancestral SARS-CoV-2 variants. WA1614G S proteins containing the six individuals S2 Omicron mutations were assessed for their fusogenicity and S surface expression after transfection in cells. Results showed that the S:N856K and N969K substitutions decreased syncytia formation and impacted S protein cell surface levels. However, we observed that “first-generation” Omicron sublineages that emerged subsequently, had convergently evolved to an enhanced fusogenic activity and S expression on the surface of infected cells while “second-generation” Omicron variants have highly diverged and showed lineage-specific fusogenic properties. Importantly, our findings could have potential implications in the improvement and redesign of COVID-19 vaccines.

Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells by miR-23b-5p-mediated activation of the Nrf2 pathway

TTo investigate the targeted regulation of the Nrf2 pathway by miR-23b-5p in transdifferentiation of rat renal tubular epithelial NRK-52E cells induced by transforming growth factor β1(TGF-β1)and the effect of Qingshen Granulesmedicated serum for alleviating transdifferentiation of NRK-52E cells. NRK-52E cells with TGF-β1-induced transdifferentiation were transfected with miR-23b-5p mimic, miR-23b-5p inhibitor or the negative control(NC)siRNA and then treated with of Qingshen Granules-medicated serum.CCK8 assay was used to detectthe changes in viability of NRK-52E cells.The targeting relationship between miR-23b-5p and Nrf2 was verified using a dual luciferase reporter gene assay.The expressions of Nrf2, Keap1 and α-SMA mRNAs and proteins in the treated cells were detected with RT-qPCR and Western blotting, and ROS production in the cells was detected with flow cytometry. Transfection of NRK-52E cells with miR-23b-5p mimic significantly increased the expression of Nrf2 mRNA, while inhibition of miR-23b-5p obviously lowered Nrf2 mRNA in the cells.Rno-miR-23b-5p significantly down-regulated the luciferase activity of Rno-Nrf2-wt but not that of Rno-Nrf2-mu(P<0.05).Treatment with TGF-β1 significantly decreased the expressions of miR-23b-5p and Nrf2 and increased the expressions of Keap1, α-SMA and ROS in NRK-52E cells(P<0.05), and these changes were obviously ameliorated by treatment with 20% Qingshen Granules-medicated serum for 24 h.Transfection of the cells with miR-23b-mimic significantly decreased the expressions of Keap1, α-SMA and ROS(P<0.05), which were further decreased by treatment with the medicated serum(P<0.05). Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells via miR-23b-5pmediated activation of the Nrf2 pathway.

Activation of PI3K/AKT/mTOR signaling axis by UBE2S inhibits autophagy leading to cisplatin resistance in ovarian cancer

BackgroundEpithelial ovarian cancer (OC) is the fourth leading cause of cancer-related deaths in women, with a 5-year survival rate of 30%-50%. Platinum resistance is the chief culprit for the high recurrence and mortality rates. Several studies confirm that the metabolic regulation of ubiquitinating enzymes plays a vital role in platinum resistance in OC.MethodsIn this study, we selected ubiquitin-conjugating enzyme E2S (UBE2S) as the candidate gene for validation. The levels of UBE2S expression were investigated using TCGA, GTEx, UALCAN, and HPA databases. In addition, the correlation between UBE2S and platinum resistance in OC was analyzed using data from TCGA. Cisplatin-resistant OC cell lines were generated and UBE2S was knocked down; the transfection efficiency was verified. Subsequently, the effects of knockdown of UBE2S on the proliferation and migration of cisplatin-resistant OC cells were examined through the CCK8, Ki-67 immunofluorescence, clone formation, wound healing, and transwell assays. In addition, the UBE2S gene was also validated in vivo by xenograft models in nude mice. Finally, the relationship between the UBE2S gene and autophagy and the possible underlying regulatory mechanism was preliminarily investigated through MDC and GFP-LC3-B autophagy detection and western blotting experiments. Most importantly, experimental validation of mTOR agonist reversion (the rescuse experiments) was also performed.ResultsUBE2S was highly expressed in OC at both nucleic acid and protein levels. The results of immunohistochemistry showed that the level of UBE2S expression in platinum-resistant samples was significantly higher relative to the platinum-sensitive samples. By cell transfection experiments, knocking down of the UBE2S gene was found to inhibit the proliferation and migration of cisplatin-resistant OC cells. Moreover, the UBE2S gene could inhibit autophagy by activating the PI3K/AKT/mTOR signaling pathway to induce cisplatin resistance in OC in vivo and in vitro.ConclusionIn conclusion, we discovered a novel oncogene, UBE2S, which was associated with platinum response in OC, and examined its key role through bioinformatics and preliminary experiments. The findings may open up a new avenue for the evaluation and treatment of OC patients at high risk of cisplatin resistance.

A Replication-Competent Retroviral Vector Expressing the HERV-W Envelope Glycoprotein is a Potential Tool for Cancer Gene Therapy.

The fusogenic membrane glycoprotein (FMG) derived from the human endogenous retrovirus-W (HERV-W) exhibits fusogenic properties, making it a promising candidate for cancer gene therapy. When cells are transfected with HERV-W FMG, they can fuse with neighboring cells expressing the receptor, resulting in the formation of syncytia. These syncytia eventually undergo cell death within a few days. In addition, it has been observed that an HERV-W env mutant, which is truncated after amino acid 483, displays increased fusogenicity compared to the wild-type HERV-W env. In this study, we observed syncytium formation upon transfection of HeLa and TE671 human cancer cells with plasmids containing the HERV-W 483 gene. To explore the potential of a semi-replication-competent retroviral (s-RCR) vector encoding HERV-W 483 for FMG-mediated cancer gene therapy, we developed two replication-defective retroviral vectors: a gag-pol vector encoding HERV-W 483 (MoMLV-HERV-W 483) and an env vector encoding VSV-G (pCLXSN-VSV-G-EGFP). When MoMLV-HERV-W 483 and pCLXSN-VSV-G-EGFP were co-transfected into HEK293T cells to produce the s-RCR vector, gradual syncytium formation was observed. However, the titers of the s-RCR virus remained consistently low. To enhance gene transfer efficiency, we constructed an RCR vector encoding HERV-W 483 (MoMLV-10A1-HERV-W 483), which demonstrated replication ability in HEK293T cells. Infection of A549 and HT1080 human cancer cell lines with this RCR vector induced syncytium formation and subsequent cell death. Consequently, both the s-RCR vector and RCR encoding HERV-W 483 hold promise as valuable tools for cancer gene therapy.

Angiotensin 1-7 Exerts Antioxidant Effects, Suppresses Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Apoptosis in Renal Proximal Tubular Cells

Oxidative stress is one of the crucial pathogenic factors involved in the progression of renal injury. Angiotensin (ANG) 1-7, a bioactive heptapeptide of the renin-angiotensin-aldosterone system is known to exert antioxidant and nephroprotective effects. However, the cellular mechanism involved in the beneficial effect of ANG 1-7 is not clear. Here, we assessed ANG 1-7’s effect on H2O2-mediated oxidative damage in the human proximal tubular (HK2) cells and the underlying mechanisms. HK2 cells were incubated with H2O2 (500µM, 4h) pre-treated with and without ANG 1-7 (100nM, 24h), and reactive oxygen species (ROS) generation, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, apoptosis and mammalian target of rapamycin (mTOR) signaling were determined. H2O2 induced an increase in oxidative and ER stress together with loss of mitochondrial membrane potential, decreased ATP levels, and induced apoptosis in HK2 cells. Moreover, H2O2 treatment resulted in the activation of mTOR complexes (mTORC1 and mTORC2) in these cells. ANG 1-7 significantly attenuated H2O2-induced ROS generation, ER stress and apoptosis, and also improved mitochondrial function. Additionally, pre-treatment of ANG 1-7 inhibited the H2O2-mediated mTOR activation. These effects of ANG 1-7 were blocked by co-treatment with the Mas receptor (MasR) inhibitor, A779. Furthermore, transfection of HK2 cells with Mas receptor siRNA also abolished the inhibitory effect of ANG 1-7 on mTOR activities. In conclusion, ANG 1-7 via MasR mitigates oxidative stress, suppresses mTOR signaling, and protects HK2 cells from ER stress, mitochondrial dysfunction, and apoptosis, suggesting ANG 1-7-MasR renoprotective effects.

ARV-825 Showed Antitumor Activity against BRD4-NUT Fusion Protein by Targeting the BRD4.

The bromodomain-containing 4 (BRD4) is a member of the bromodomain and extra terminal domain (BET) family, which is an important epigenetic reader. It is currently a promising oncology target. In some tumors, BET bromodomain inhibitors have demonstrated promising results. Proteolysis-targeting methods (PROTAC), which rapidly and effectively degrade BRD4, have displayed considerable potential in the treatment of tumors in recent years. The purpose of this study is to examine the potential impact of BRD4 PROTAC compounds ARV-825 on oncogene BRD4-NUT fused protein in NUT carcinoma. The effectiveness of ARV-825 was evaluated at the cellular level using the cell counting kit 8 test, wound healing, cell transfection, western blotting analysis, and RNA sequencing. The effectiveness of ARV-825 was also examined in vivo using a xenograft model. The BRD4-NUT fusion gene was overexpressed in 3T3 cells, and the pathogenic fusion gene was simulated. The results showed that the overexpression of BRD4-NUT could promote the proliferation and migration of 3T3 cells, but the expression of BRD4 protein was degraded after the addition of the novel cereblon-based PROTAC compound ARV-825 against BRD4, resulting in inhibition of BRD4-NUT 3T3 cell proliferation and migration. Further RNA-seq analysis showed that overexpression of BRD4-NUT was accompanied by increased expression of gene (e.g., Myc, E2F, TRAFs, Wnt, Gadd45g, and Sox6) with significantly enriched pathway (e.g., small cell lung cancer, NF-kappa B signaling pathway, and breast cancer), promoted cell cycle from G 1 phase to S phase, and increased cell proliferation and migration, activated the antiapoptosisi signal, led to abnormal cell growth, and ultimately led to tumorigenesis. The addition of ARV-825 effectively rescued this process and effectively inhibited cell vitality, proliferation, and migration. In vivo studies demonstrated that treatment with ARV-825 greatly suppressed tumor growth without causing harmful side effects and downregulated the BRD4-NUT expression level. Through the induction of BRD4 protein degradation, ARV-825 can successfully limit BRD4-NUT 3T3 cell proliferation in vitro and in vivo. These findings suggested that the BRD4 inhibitor ARV-825 would be an effective therapeutic strategy for treating NUT carcinoma that with the genetic feature of BRD4-NUT fusion event.

An antibody-free evaluation of an mRNA COVID-19 vaccine

This manuscript describes the use of an analytical assay that combines transfection of mammalian cells and isotope dilution mass spectrometry (IDMS) for accurate quantification of antigen expression. Expired mRNA COVID-19 vaccine material was stored at 4 °C, room temperature (∼25 °C), and 56 °C over a period of 5 weeks. The same vaccine was also exposed to 5 freeze-thaw cycles. Every week, the spike protein antigenic expression in mammalian (BHK-21) cells was evaluated. Housekeeping proteins, β-actin and GAPDH, were simultaneously quantified to account for the variation in cell counts that occurs during maintenance and growth of cell cultures. Data show that vaccine stored at elevated temperatures results in reduced spike protein expression. Also, maintaining the vaccine in ultracold conditions or exposing the vaccine to freeze-thaw cycles had less effect on the vaccine's ability to produce the antigen in mammalian cells. We describe the use of IDMS as an antibody-free means to accurately quantify expressed protein from mammalian cells transfected with mRNA vaccine.

Paradoxical increase of neurofilaments in SMA patients treated with onasemnogene abeparvovec-xioi.

Neurofilament light chain (NfL) has been proposed as a biomarker reflecting disease severity and therapy response in children with spinal muscular atrophy type 1 and 2 (SMA1 and 2). The objective of this study was to examine how serum NfL changes after gene replacement therapy (GRT) with onasemnogene abeparvovec-xioi. We measured NfL in serum probes from 19 patients (10 SMA 1 and 6 SMA 2; 15 previously treated with nusinersen or risdiplam; 12 male) before and at variable time points after GRT. These values were related to motor scores (CHOP-Intend, HFMSE and RULM). Median age at GRT was 19 months (range 2-46 months). Median NfL of all patients before GRT was 39 pg/ml (range 0-663 pg/ml; normal values <25 pg/ml), increased significantly to 297 pg/ml (range 61-1,696 pg/ml; p<0,002) 1 month after GRT, and decreased to 49 pg/ml (range 24-151 pg/ml) after 6 months. Subjects pre-treated with nusinersen or risdiplam had lower baseline NfL levels than naïve patients (p<0,005), but absolute increases of NfL were similar in both groups. While motor scores were improved in 14 out of 18 SMA patients (78%) 6 months after GRT NfL values differed not significantly from those measured at baseline (p = 0,959). Serum NfL showed a paradoxical transient increase after GRT in both, pre-treated and naïve patients, which may reflect an immunological reaction in the CNS related to transfection of neuronal cells by AAV9. The clinical meaning of this increase should be assessed in future studies. Our findings encourage regular monitoring of NfL in OA treated patients.

Exploring Various Transfection Approaches and Their Applications in Studying the Regenerative Potential of Dental Pulp Stem Cells.

Transfection is a contemporary approach for introducing foreign genetic material into target cells. The effective transport of genetic materials into cells is mostly influenced by (a) the characteristics of the genetic material (quantity and quality), (b) the transfection procedure (incubation time, ratio of the reagents to the introduced genetic material, and components of cell culture), and (c) targeted cells for transfection (cell origin and cell type). This review summarizes the findings of different studies focusing on various transfection approaches and their applications to explore the regenerative potential of dental pulp stem cells (DPSCs). Several databases, including Scopus, Google Scholar, and PubMed, were searched to obtain the literature for the current review. Different keywords were used as key terms in the search. Approximately 200 articles were retained after removing duplicates from different databases. Articles published in English that discussed different transfection approaches were included. Several sources were excluded because they did not meet the inclusion criteria. Approximately 70 relevant published sources were included in the final stage to achieve the study objectives. This review demonstrated that no single transfection system is applicable to all cases and the various cell types with no side effects. Further studies are needed to focus on optimizing process parameters, decreasing the toxicity and side effects of available transfection techniques, and increasing their efficiencies. Moreover, this review sheds light on the impact of using different valuable transfection approaches to investigate the regenerative potential of DPSCs.

Construction and validation of a novel NAD+ metabolism-related risk model for prognostic prediction in osteosarcoma.

Currently, the prognosis of osteosarcoma (OS) remains discouraging, especially in elderly/metastatic OS patients. By impairing the antitumor effect of immune cells, tumor immune microenvironment (TIME) provides an environment conducive to tumor proliferation, which highly requires accelerated nicotinamide adenine dinucleotide (NAD+) metabolism for energy. Recently, many genes involved in the sustained production of NAD+ in malignant tumors have been verified to be possible prognostic indicators and therapeutic targets. Therefore, the current study was to probe into the association of NAD+ metabolism-related genes with TIME, immunotherapeutic response, and prognosis in OS. All OS data for the study were acquired from TARGET and GEO databases. In bioinformatics analysis, we performed Cox analysis, consensus clustering, principal component analysis, t-distributed stochastic neighbor embedding, uniform manifold approximation and projection, gene set enrichment analysis, gene set variation analysis, Lasso analysis, survival and ROC curves, nomogram, immune-related analysis, drug sensitivity analysis, and single-cell RNA sequencing (scRNA-seq) analysis. Cell transfection assay, RT-qPCR, western blot analysis, as well as cell wound healing, migration, and invasion assays were performed in vitro. Bioinformatics analysis identified A&B clusters and six NAD+ metabolism-related differentially expressed genes, constructed risk model and nomogram, and performed immune-related analysis, drug susceptibility analysis, and scRNA-seq analysis to inform the clinical treatment framework. In vitro experiment revealed that CBS and INPP1 can promote migration, proliferation as well as invasion of OS cells through TGF-β1/Smad2/3 pathway. Based on bioinformatics analysis and in vitro validation, this study confirmed that NAD+ metabolism affects TIME to suggest the prognosis of OS.

In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism.

With six therapies approved by the Food and Drug Association, chimeric antigen receptor (CAR) T cells have reshaped cancer immunotherapy. However, these therapies rely on ex vivo viral transduction to induce permanent CAR expression in T cells, which contributes to high production costs and long-term side effects. Thus, this work aims to develop an in vivo CAR T cell engineering platform to streamline production while using mRNA to induce transient, tunable CAR expression. Specifically, an ionizable lipid nanoparticle (LNP) is utilized as these platforms have demonstrated clinical success in nucleic acid delivery. Though LNPs often accumulate in the liver, the LNP platform used here achieves extrahepatic transfection with enhanced delivery to the spleen, and it is further modified via antibody conjugation (Ab-LNPs) to target pan-T cell markers. The in vivo evaluation of these Ab-LNPs confirms that targeting is necessary for potent T cell transfection. When using these Ab-LNPs for the delivery of CAR mRNA, antibody and dose-dependent CAR expression and cytokine release are observed along with B cell depletion of up to 90%. In all, this work conjugates antibodies to LNPs with extrahepatic tropism, evaluates pan-T cell markers, and develops Ab-LNPs capable of generating functional CAR T cells in vivo.

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios.

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

Combinatorial design of ionizable lipid nanoparticles for muscle-selective mRNA delivery with minimized off-target effects

Ionizable lipid nanoparticles (LNPs) pivotal to the success of COVID-19 mRNA (messenger RNA) vaccines hold substantial promise for expanding the landscape of mRNA-based therapies. Nevertheless, the risk of mRNA delivery to off-target tissues highlights the necessity for LNPs with enhanced tissue selectivity. The intricate nature of biological systems and inadequate knowledge of lipid structure–activity relationships emphasize the significance of high-throughput methods to produce chemically diverse lipid libraries for mRNA delivery screening. Here, we introduce a streamlined approach for the rapid design and synthesis of combinatorial libraries of biodegradable ionizable lipids. This led to the identification of iso-A11B5C1, an ionizable lipid uniquely apt for muscle-specific mRNA delivery. It manifested high transfection efficiencies in muscle tissues, while significantly diminishing off-targeting in organs like the liver and spleen. Moreover, iso-A11B5C1 also exhibited reduced mRNA transfection potency in lymph nodes and antigen-presenting cells, prompting investigation into the influence of direct immune cell transfection via LNPs on mRNA vaccine effectiveness. In comparison with SM-102, while iso-A11B5C1’s limited immune transfection attenuated its ability to elicit humoral immunity, it remained highly effective in triggering cellular immune responses after intramuscular administration, which is further corroborated by its strong therapeutic performance as cancer vaccine in a melanoma model. Collectively, our study not only enriches the high-throughput toolkit for generating tissue-specific ionizable lipids but also encourages a reassessment of prevailing paradigms in mRNA vaccine design. This study encourages rethinking of mRNA vaccine design principles, suggesting that achieving high immune cell transfection might not be the sole criterion for developing effective mRNA vaccines.

HACE1 expression in heart failure patients might promote mitochondrial oxidative stress and ferroptosis by targeting NRF2.

Heart failure is a prevalent and life-threatening medical condition characterized by abnormal atrial electrical activity, contributing to a higher risk of ischemic stroke. Atrial remodelling, driven by oxidative stress and structural changes, plays a central role in heart failure progression. Recent studies suggest that HACE1, a regulatory gene, may be involved in cardiac protection against heart failure. Clinical data analysis involved heart failure patients, while an animal model utilized C57BL/6J mice. RT-PCR, microarray analysis, histological examination, ELISA, and Western blot assays were employed to assess gene and protein expression, oxidative stress, and cardiac function. Cell transfection and culture of mouse atrial fibroblasts were performed for in-vitro experiments. HACE1 expression was reduced in heart failure patients and correlated negatively with collagen levels. In mouse models, HACE1 up-regulation reduced oxidative stress, mitigated fibrosis, and improved cardiac function. Conversely, HACE1 knockdown exacerbated oxidative stress, fibrosis, and cardiac dysfunction. HACE1 also protected against ferroptosis and mitochondrial damage. NRF2, a transcription factor implicated in oxidative stress, was identified as a target of HACE1, with HACE1 promoting NRF2 activity through ubiquitination. HACE1 emerges as a potential therapeutic target and diagnostic marker for heart failure. It regulates oxidative stress, mitigates cardiac fibrosis, and protects against ferroptosis and mitochondrial damage. The study reveals that HACE1 achieves these effects, at least in part, through NRF2 activation via ubiquitination, offering insights into novel mechanisms for heart failure pathogenesis and potential interventions.

Lipoamino bundle LNPs for efficient mRNA transfection of dendritic cells and macrophages show high spleen selectivity

Messenger RNA (mRNA) is a powerful tool for nucleic acid-based therapies and vaccination, but efficient and specific delivery to target tissues remains a significant challenge. In this study, we demonstrate lipoamino xenopeptide carriers as components of highly efficient mRNA LNPs. These lipo-xenopeptides are defined as 2D sequences in different 3D topologies (bundles or different U-shapes). The polar artificial amino acid tetraethylene pentamino succinic acid (Stp) and various lipophilic tertiary lipoamino fatty acids (LAFs) act as ionizable amphiphilic units, connected in different ratios via bisamidated lysines as branching units. A series of more lipophilic LAF4-Stp1 carriers with bundle topology is especially well suited for efficient encapsulation of mRNA into LNPs, facilitated cellular uptake and strongly enhanced endosomal escape. These LNPs display improved, faster transfection kinetics compared to standard LNP formulations, with high potency in a variety of tumor cell lines (including N2a neuroblastoma, HepG2 and Huh7 hepatocellular, and HeLa cervical carcinoma cells), J774A.1 macrophages, and DC2.4 dendritic cells. High transfection levels were obtained even in the presence of serum at very low sub-microgram mRNA doses. Upon intravenous application of only 3 µg mRNA per mouse, in vivo mRNA expression is found with a high selectivity for dendritic cells and macrophages, resulting in a particularly high overall preferred expression in the spleen.

In Situ Stability Test for mRNA Vaccines Based on Deep-UV Resonance Raman Spectroscopy.

Deep-UV resonance Raman spectroscopy has been shown to offer great potential for probing the in situ stability of mRNA vaccines. In this study, a vaccine model was subjected to controlled degradation using RNase A or through aging at room temperature. The degradation of mRNA was confirmed by using a cell transfection test and by gel electrophoresis. Under both settings, DUVRR spectroscopy successfully revealed the mRNA degradation signs of the vaccine model.