Suitable Promoter for DNA Vaccination Using a pDNA Ternary Complex.

In this study, we evaluated the effect of several promoters on the transfection activity and immune-induction efficiency of a plasmid DNA (pDNA)/polyethylenimine/γ-polyglutamic acid complex (pDNA ternary complex). Model pDNAs encoding firefly luciferase (Luc) were constructed with several promoters, such as simian virus 40 (SV40), eukaryotic elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and chicken beta actin hybrid (CBh) (pSV40-Luc, pEF1-Luc, pCMV-Luc, and pCBh-Luc, respectively). Four types of pDNA ternary complexes, each with approximately 145-nm particle size and -30-mV ζ-potential, were stably constructed. The pDNA ternary complex containing pSV40-Luc showed low gene expression, but the other complexes containing pEF1-Luc, pCMV-Luc, and pCBh-Luc showed high gene expression in DC2.4 cells and spleen after intravenous administration. After immunization using various pDNA encoding ovalbumin (OVA) such as pEF1-OVA, pCMV-OVA, and pCBh-OVA, only the pDNA ternary complex containing pCBh-OVA showed significant anti-OVA immunoglobulin G (IgG) induction. In conclusion, our results showed that the CBh promoter is potentially suitable for use in pDNA ternary complex-based DNA vaccination.

Effect of a novel siRNA delivery system, siRNA ternary complex, on melanoma lung metastasis.

In this study, we determined effects of an anionic siRNA delivery vector, siRNA ternary complex, which is constructed with biodegradable dendrigraft poly-L-lysine (DGL) and γ-polyglutamic acid (γ-PGA) on the melanoma cells and melanoma lung metastasis. The siRNA ternary complex showed high cellular uptake and silencing effect in melanoma cell line B16-F10/Luc cells. After intravenous administration of the siRNA ternary complex, high silencing effect was also observed in the lung of B16-F10/Luc melanoma metastasis model mice. Therefore, we applied vascular endothelial growth factor (VEGF)-siRNA on the siRNA ternary complex and determined the effect on the melanoma lung metastasis. The siRNA ternary complex containing VEGF-siRNA reduced VEGF protein levels significantly in in vitro and in vivo, and the complex successfully inhibited melanoma lung metastasis. This biodegradable and effective siRNA delivery vector, siRNA ternary complex, could be available for clinical trials.

Effect of rifampicin administration on CYP induction in a dermatomyositis patient with vasospastic angina attributable to nilmatrelvir/ritonavir-induced blood tacrolimus elevation: A case report.

Ritonavir (RTV), which is used in combination with nilmatrelvir (NMV) to treat coronavirus disease 2019 (COVID-19), inhibits cytochrome P450 (CYP) 3A, thereby increasing blood tacrolimus (TAC) levels through a drug-drug interaction (DDI). We experienced a case in which a DDI between the two drugs led to markedly increased blood TAC levels, resulting in vasospastic angina (VSA) and acute kidney injury (AKI). Rifampicin (RFP) was administered to induce CYP3A and promote TAC metabolism. A 60-year-old man with dermatomyositis who was taking 3 mg/day TAC contracted COVID-19. The patient started oral NMV/RTV therapy, and he was admitted to the hospital after 4 days because of chest pain and AKI. On day 5, his blood TAC level increased markedly to 119.8 ng/mL. RFP 600 mg was administered once daily for 3 days, and his blood TAC level decreased to the therapeutic range of 9.6 ng/mL on day 9, leading to AKI improvement. Transient complete atrioventricular block and nonsustained ventricular tachycardia were present during chest pain. In the coronary spasm provocation test, complete occlusion was observed in the right coronary artery, leading to a diagnosis of VSA. VSA and AKI are possible side effects of high blood TAC levels caused by DDI, and attention should be paid to cardiovascular side effects such as VSA and AKI associated with increased blood levels of TAC when it is used together with NMV/RTV. When blood levels of TAC increase, oral RFP can rapidly decrease TAC blood levels and potentially reduce its toxicity.

Effects of changing the timing of warfarin administration in combination with fluconazole on prolongation of the PT-INR: a case report.

BACKGROUND: Fluconazole (FLCZ) inhibits cytochrome P450 (CYP) 2C9, 2C19, and 3A4 and has a drug-drug interaction that potentiates the effects of warfarin and prolong the prothrombin time-international normalized ratio (PT-INR). Although a drug-drug interaction have been reported between FLCZ and warfarin, the effects of the timing of their administration on this interaction have not yet been investigated. CASE PRESENTATION: A female patient in her 30s with Marfan syndrome had undergone the Bentall procedure with a mechanical valve and total arch replacement for acute aortic dissection Stanford A type and rupture of the ascending aorta. Warfarin was administered to prevent thromboembolism. She was hospitalized 1 year ago for graft infection caused by Candida albicans, and treatment with FLCZ was initiated. She received FLCZ 200 mg once a day in the morning and warfarin 1.75 mg once a day in the evening, and the PT-INR remained stable at approximately 2.0 and within the therapeutic range. However, 42 days after changing the timing of administration of warfarin from evening to morning, the PT-INR was prolonged by approximately 3-fold to 6.25. The PT-INR then decreased to the previous level by changing the timing of administration of warfarin from morning to evening. CONCLUSIONS: The timing of administration of FLCZ and warfarin may affect the magnitude of drug-drug interaction.

Poor Renal Function and a High Modified Glasgow Prognostic Score Are Predictive Factors for Nedaplatin/5-Fluorouracil Combination Therapy-induced Febrile Neutropenia.

BACKGROUND/AIM: Nedaplatin (NDP)/5-fluorouracil (5-FU) combination therapy frequently causes severe neutropenia and febrile neutropenia (FN). However, there is no consensus on the risk factors for FN caused by NDP/5-FU combination therapy. Mouse models of cancer cachexia are known to be susceptible to infections. Conversely, the modified Glasgow prognostic score (mGPS) is believed to reflect cancer cachexia. We hypothesized that mGPS is a predictive factor for FN caused by NDP/5-FU combination therapy. PATIENTS AND METHODS: We analyzed the relationship between mGPS and FN in patients who received NDP/5-FU combination therapy at Nagasaki University Hospital using multivariate logistic analysis. RESULTS: In total, 157 patients were studied, 20 of whom developed FN (12.7%). Multivariate analysis revealed that mGPS 1-2 [odds ratio (OR)=4.13, 95% confidence interval (CI)=1.42-12.02, p=0.009] and creatinine clearance <54.4 ml/min (OR=5.81, 95% CI=1.81-18.59, p=0.003) were significantly associated with the development of FN. CONCLUSION: Several guidelines suggest that patients receiving chemotherapy with an FN rate 10-20% should be considered for prophylactic granulocyte colony-stimulating factor (G-CSF), depending on the individual patient's risk of developing FN. When NDP/5-FU combination therapy is administered to patients with risk factors identified in this study, prophylactic administration of G-CSF should be considered. In addition, the neutrophil count and axillary temperature should be monitored more frequently.

Influence of lipid composition of messenger RNA-loaded lipid nanoparticles on the protein expression via intratracheal administration in mice.

Intratracheal (i.t.) administration, which takes advantage of the specific structure of the respiratory system, can effectively deliver nanoparticles to the lung. Much remains unknown about the i.t. administration of messenger RNA (mRNA)-lipid nanoparticles (LNPs) and the effect of lipid composition. In this study, we administered minute amounts of mRNA-LNP solutions into mice intratracheally and investigated the effect of lipid composition on protein expression in the lungs. We first validated higher protein expression with mRNA-LNP compared to that with mRNA-PEI complex and naked mRNA. Then, we evaluated the influence of lipid composition of LNPs on the protein expression and found that: 1) decreasing the PEG molarity from 1.5% to 0.5% could significantly increase the protein expression; 2) replacing DMG-PEG with DSG-PEG could slightly increase the protein expression; 3) using DOPE instead of DSPC could increase protein expression by an order of magnitude. We successfully prepared an mRNA-LNP with optimal lipid compositions that led to robust protein expression following i.t. administration, thus providing meaningful insights into advanced development of mRNA-LNPs for therapeutic i.t. administration.

Immune complexome analysis of a rich variety of serum immune complexes identifies disease-characteristic immune complex antigens in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular endothelial dysfunction and skin fibrosis. Recently, the presence and pathogenic role of immune complexes (ICs) of SSc patients were reported. However, the identities of antigens in these ICs are unknown. Therefore, we examined ICs in the serum of SSc patients to elucidate SSc pathogenesis. In this study, IC concentrations in serum samples from SSc and systemic lupus erythematosus (SLE) patients were measured by C1q enzyme-linked immunosorbent assays; immune complex analysis was used for comprehensive identification and comparison of antigens incorporated into ICs (IC-antigens). The expression patterns of SSc-specific IC-antigens in skin sections were investigated by immunohistochemistry. Compared with SLE patients who developed disease because of IC deposition, SSc patients had a greater number of IC-antigens and a smaller difference in IC concentrations, suggesting that SSc pathogenesis is affected by the proteins present in ICs. In contrast, the IC concentration and number of IC-antigens did not significantly differ according to the clinical phenotype of SSc. We identified 478 IC-antigens in SSc patients, including multiple RNAP II-associated proteins that were targeted by antibodies previously associated with SSc pathogenesis. The most frequently detected RNAP II-associated protein, RNA polymerase II transcription subunit 30 (MED30), was strongly expressed at lesion sites and reportedly regulates endothelial differentiation. Therefore, increased expression of MED30 in lesions may have an antigenic effect, and MED30 function may be impaired or inhibited by IC formation. RNAP II-associated proteins may SSc pathogenesis through mechanisms such as the MED30 pathway.

Examination of the Effect of Proton Pump Inhibitors on the Anticancer Activity of Oxaliplatin.

BACKGROUND/AIM: Oxaliplatin (L-OHP) is absorbed by cancer cells via organic cation transporter1-3 (OCT1-3). However, proton pump inhibitors (PPIs) suppress the function of OCT1-3. This study investigated whether PPIs attenuate the antitumor effect of L-OHP. PATIENTS AND METHODS: Colorectal cancer patients who received FOLFOX (L-OHP + 5-fluorouracil: 5-FU) + bevacizumab therapy at Nagasaki University Hospital from October 1, 2010 to September 30, 2019 were retrospectively investigated. Patients were categorized into two groups with or without PPIs use. Progression-free survival (PFS) between the two groups was compared using the log-rank test. L-OHP was added to the intestinal epithelial Caco-2 cell line with or without the PPI rabeprazole, and then cell viability was analyzed using the WST-8 cell proliferation assay. RESULTS: The median PFS was 11.4 months in the group with PPIs and 9.7 months in the group without PPIs (p=0.736). No significant effect of 1-10 µM rabeprazole was observed on the antitumor effect of L-OHP. Plasma concentrations of rabeprazole at clinical doses are 1.0-1.3 µM. CONCLUSION: Even if L-OHP interacts with PPIs, clinical doses of PPIs were considered to have minimal effect on the antitumor effect of L-OHP.

[Wasted Anti-Cancer Agents Due to Cancellations after Dispensing].

At our hospital, anti-cancer drug administration is managed using a regimen-ordering system, and orders for the outpatient department and hospital wards have to be placed by 15:00 and 14:00 the day before they are required. On the day of treatment, the doctor examines the patient, confirms the test results, and places the final order for treatment on the patient's electronic medical record. In response, the pharmacist adjusts the anti-cancer drug preparation, and treatment is provided in the outpatient setting or in a ward. Although drug costs have increased due to the widespread use of immunotherapy, there have been cases where a drug was wasted after the required amount was adjusted on the day of treatment or drugs were discarded altogether, which pose serious problems. From April 2016 to March 2021, the total number of cases of drug wastage following placement of the final treatment order and drug disposal were 146 and 84, respectively, and the total associated economic loss was 5.81 million yen. The main causes were pre-confirmation mistakes and patients' physical condition on the day of treatment; some cancellations caused by patient-related factors were unavoidable. The current status of drug disposal is reported to the hospital director every 6 months, and the doctor-in-charge is interviewed regarding the reason for the wastage. In cases involving the disposal of large quantities of drugs(≥100,000 yen), the department manager and medical office manager are contacted, and an incident report is submitted. In 2021, drugs worth 2.03 million yen were discarded between April and September, which is worth serious consideration. It is essential to understand the reasons for drug wastage, pay attention to expensive regimens, and take appropriate measures at each facility.

Immune complexome analysis of serum samples from non-small-cell lung cancer patients identifies predictive biomarkers for nivolumab therapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have achieved important outcomes in cancer treatment. However, current clinical biomarker tests are not suitable for some patients because they require tumor tissues and have poor predictive value for treatment responses. Therefore, the identification of biomarkers that enable screening tests in all patients is necessary. METHODS: We performed an immune complexome analysis of non-small cell lung cancer patients treated with nivolumab to comprehensively identify and compare antigens incorporated into immune complexes (IC-antigens) in serum samples from the responders (n = 15) and non-responders (n = 20). Additionally, combinations of IC-antigens characteristic to the responder group were evaluated by logistic regression analysis and receiver operating characteristics curves to examine their predictiveness for ICI treatment responses. RESULTS: The combination of predictive biomarkers detected before treatment was profilin-1, purine nucleoside phosphorylase, alpha-enolase, and nucleoside diphosphate kinase A [p = 0.0043, odds ratio = 2.26, 95% confidence interval (CI) = 1.19-4.28, area under the curve = 0.76]. The combination of predictive biomarkers detected after treatment was peptidyl-prolyl cis-trans isomerase A, ubiquitin-like modifier-activating enzyme 1, complement component C8 beta chain, and apolipoprotein L1 (p = 0.0039, odds ratio = 2.56, 95% CI = 1.25-5.23, area under the curve = 0.77). CONCLUSION: Combinations of serum IC-antigens may predict the therapeutic effect of nivolumab in non-small cell lung cancer patients.

Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation.

We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and ß-tricalcium phosphate (ß-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 µg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 µg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

Delivery of pDNA to the Lung by Lipopolyplexes Using N-Lauroylsarcosine and Effect on the Pulmonary Fibrosis.

In a previous study, we constructed a lung-targeting lipopolyplex containing polyethyleneimine (PEI), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and N-lauroylsarcosine (LS). The lipopolyplex exhibited an extremely high gene expression in the lung after intravenous administration. Here, we optimized the lipopolyplex and used it to deliver a TGF-ß1 shRNA to treat refractory pulmonary fibrosis. We constructed several lipopolyplexes with pDNA, various cationic polymers, cationic lipids, and LS to select the most effective formulation. Then, the pDNA encoding shRNA against mouse TGF-ß1 was encapsulated in the lipopolyplex and injected into mice with bleomycin-induced pulmonary fibrosis. After optimizing the lipopolyplex, dendrigraft poly-L-lysine (DGL) and DOTMA were selected as the appropriate cationic polymer and lipid, respectively. The lipopolyplex was constructed with a pDNA, DGL, DOTMA, and LS charge ratio of 1:2:2:4 showed the highest gene expression. After intravenous administration of the lipopolyplex, the highest gene expression was observed in the lung. In the in vitro experiment, the lipopolyplex delivered pDNA into the cells via endocytosis. As a result, the lipopolyplex containing pDNA encoding TGF-ß1 shRNA significantly decreased hydroxyproline in the pulmonary fibrosis model mice. We have successfully inhibited pulmonary fibrosis using a novel lung-targeting lipopolyplex.

Analysis of Predictive Factors for Diarrhea after the Administration of Naldemedine.

Naldemedine (NAL), a peripherally acting µ-opioid receptor antagonist, is effective for opioid-induced constipation (OIC). However, diarrhea is the most common adverse event. We investigated the incidence of NAL-induced diarrhea in patients who started NAL at Nagasaki University Hospital between June 2017 and March 2019. Predictors of NAL-induced diarrhea were analyzed using a multivariate logistic regression model. Two hundred and forty-two patients were included in the present study, and NAL-induced diarrhea was observed in 17.8% (43 patients). The results of multiple logistic regression analyses identified the administration of opioid analgesics for 8 d or longer before the initiation of NAL (odds ratio (OR): 2.20, 95% confidence interval (95% CI): 1.04-4.64, p = 0.039), the combination of a laxative (OR: 2.22, 95%CI: 1.03-4.81, p = 0.042), and the combination of CYP3A4 inhibitors (strong/moderate) (OR: 2.80, 95%CI: 1.02-7.67, p = 0.045) as risk factors. Therefore, the development of diarrhea needs to be considered in patients with these risk factors. Furthermore, diarrhea may be controlled by the initiation of NAL within 7 d of opioid analgesics and, where possible, the discontinuation of or change in the combination of moderate or strong CYP3A4 inhibitors.

Induction of mucosal immunity by pulmonary administration of a cell-targeting nanoparticle.

We previously found that a nanoparticle constructed with an antigen, benzalkonium chloride (BK) and γ-polyglutamic acid (γ-PGA) showed high Th1 and Th2-type immune induction after subcutaneous administration. For prophylaxis of respiratory infections, however, mucosal immunity should be induced. In this study, we investigated the effect of pulmonary administration of a nanoparticle comprising ovalbumin (OVA) as a model antigen, BK, and γ-PGA on induction of mucosal immunity in the lungs and serum. The complex was strongly taken up by RAW264.7 and DC2.4cells. After pulmonary administration, lung retention was longer for the OVA/BK/γ-PGA complex than for OVA alone. OVA-specific serum immunoglobulin (Ig)G was highly induced by the complex. High IgG and IgA levels were also induced in the bronchoalveolar lavage fluid, and in vivo toxicities were not observed. In conclusion, we effectively and safely induced mucosal immunity by pulmonary administration of an OVA/BK/γ-PGA complex.

Mesenchymal stem cell-derived extracellular vesicles as probable triggers of radiation-induced heart disease.

BACKGROUND: Radiation-induced heart disease has been reported, but the underlying mechanisms remain unclear. Mesenchymal stem cells (MSCs), also residing in the heart, are highly susceptible to radiation. We examined the hypothesis that the altered secretion of extracellular vesicles (EVs) from MSCs is the trigger of radiation-induced heart disease. METHODS: By exposing human placental tissue-derived MSCs to 5 Gy γ-rays, we then isolated EVs from the culture medium 48 h later and evaluated the changes in quantity and quality of EVs from MSCs after radiation exposure. The biological effects of EVs from irradiated MSCs on HUVECs and H9c2 cells were also examined. RESULTS: Although the amount and size distribution of EVs did not differ between the nonirradiated and irradiated MSCs, miRNA sequences indicated many upregulated or downregulated miRNAs in irradiated MSCs EVs. In vitro experiments using HUVEC and H9c2 cells showed that irradiated MSC-EVs decreased cell proliferation (P < 0.01), but increased cell apoptosis and DNA damage. Moreover, irradiated MSC-EVs impaired the HUVEC tube formation and induced calcium overload in H9c2 cells. CONCLUSIONS: EVs released from irradiated MSCs show altered miRNA profiles and harmful effects on heart cells, which provides new insight into the mechanism of radiation-related heart disease risks.

Gene-activated matrix harboring a miR20a-expressing plasmid promotes rat cranial bone augmentation.

Gene-activated matrix (GAM) has a potential usefulness in bone engineering as an alternate strategy for the lasting release of osteogenic proteins but efficient methods to generate non-viral GAM remain to be established. In this study, we investigated whether an atelocollagen-based GAM containing naked-plasmid (p) DNAs encoding microRNA (miR) 20a, which may promote osteogenesis in vivo via multiple pathways associated with the osteogenic differentiation of mesenchymal stem/progenitor cells (MSCs), facilitates rat cranial bone augmentation. First, we confirmed the osteoblastic differentiation functions of generated pDNA encoding miR20a (pmiR20a) in vitro, and its transfection regulated the expression of several of target genes, such as Bambi1 and PPARγ, in rat bone marrow MSCs and induced the increased expression of BMP4. Then, when GAMs fabricated by mixing 100 µl of 2% bovine atelocollagen, 20 mg ß-TCP granules and 0.5 mg (3.3 µg/µl) AcGFP plasmid-vectors encoding miR20a were transplanted to rat cranial bone surface, the promoted vertical bone augmentation was clearly recognized up to 8 weeks after transplantation, as were upregulation of VEGFs and BMP4 expressions at the early stages of transplantation. Thus, GAM-based miR delivery may provide an alternative non-viral approach by improving transgene efficacy via a small sequence that can regulate the multiple pathways.

Evaluation of transgene expression characteristics and DNA vaccination against melanoma metastasis of an intravenously injected ternary complex with biodegradable dendrigraft poly-L-lysine in mice.

We developed a biocompatible splenic vector for a DNA vaccine against melanoma. The splenic vector is a ternary complex composed of plasmid DNA (pDNA), biodegradable dendrigraft poly-L-lysine (DGL), and γ-polyglutamic acid (γ-PGA), the selective uptake of which by the spleen has already been demonstrated. The ternary complex containing pDNA encoding luciferase (pCMV-Luc) exhibited stronger luciferase activity for RAW264.7 mouse macrophage-like cells than naked pCMV-Luc. Although the ternary complex exhibited strong luciferase activity in the spleen after its tail vein injection, luciferase activity in the liver and spleen was significantly decreased by a pretreatment with clodronate liposomes, which depleted macrophages in the liver and spleen. These results indicate that the ternary complex is mainly transfected in macrophages and is a suitable formulation for DNA vaccination. We applied the ternary complex to a pUb-M melanoma DNA vaccine. The ternary complex containing pUb-M suppressed the growth of melanoma and lung metastasis by B16-F10 mouse melanoma cells. We also examined the acute and liver toxicities of the pUb-M ternary complex at an excess pDNA dose in mice. All mice survived the injection of the excess amount of the ternary complex. Liver toxicity was negligible in mice injected with the excess amount of the ternary complex. In conclusion, we herein confirmed that the ternary complex was mainly transfected into macrophages in the spleen after its tail vein injection. We also showed the prevention of melanoma metastasis by the DNA vaccine and the safety of the ternary complex.

Anionic Complex with Efficient Expression and Good Safety Profile for mRNA Delivery.

We previously found that a complex comprising plasmid DNA (pDNA), polyethylenimine (PEI), and γ-polyglutamic acid (γ-PGA) had high transgene efficiency without cytotoxicity in vitro and in vivo. However, messenger RNA (mRNA) remains an attractive alternative to pDNA. In this study, we developed a safe and effective delivery system for mRNA to prevent its degradation and efficiently deliver it into target cells. Various cationic and anionic complexes were produced containing PEI, γ-PGA, and an mRNA encoding firefly luciferase. Their physicochemical properties and cytotoxicities were analyzed and the in vitro and in vivo protein expression were determined. The cationic mRNA/PEI complex showed high in vitro protein expression with strong cytotoxicity. The anionic complex was constructed as mRNA/PEI8/γ-PGA12 complex with a theoretical charge ratio of 1:8:12 based on the phosphate groups of the mRNA, nitrogen groups of PEI, and carboxylate groups of γ-PGA. It was stable and showed high in vitro protein expression without cytotoxicity. After intravenous administration of mRNA/PEI8/γ-PGA12 complex to mice, high protein expression was observed in the spleen and liver and slight expression was observed in the lung over 24 h. Thus, the newly constructed mRNA/PEI8/γ-PGA12 complex provides a safe and effective strategy for the delivery of mRNA.

Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues.

The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and γ-polyglutamic acid (γ-PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination.

Proliferation-associated long noncoding RNA, TMPO-AS1, is a potential therapeutic target for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer compared with luminal or epidermal growth factor receptor 2 subtypes, thus effective therapeutic options for TNBC are yet to be developed. Nowadays, oncogenic long noncoding RNAs (lncRNAs) are applied to cancer management as a new class of therapeutic targets. We previously showed that thymopoietin antisense transcript 1 (TMPO-AS1) is a proliferation-associated lncRNA that contributes to hormone-dependent breast cancer progression by stabilizing estrogen receptor-α mRNA. We here showed that TMPO-AS1 is abundantly expressed in basal-like breast cancer subtype based on the transcriptomic data in The Cancer Genome Atlas as well as in TNBC cell lines and patient-derived cells. Small interfering RNA-based loss-of-function analyses showed that TMPO-AS1 knockdown substantially represses the proliferation and migration of TNBC cells. Expression microarray analysis showed that TMPO-AS1 alters gene signatures related to transforming growth factor-ß signaling in addition to proliferative E2F signaling pathways. TMPO-AS1-targeted siRNA treatment through engineered drug delivery systems using cancer-targeted polyion complex micelle or nanoball technology significantly impaired the in vivo growth of primary and metastatic TNBC xenograft tumors. Our findings suggest that TMPO-AS1 plays a key role in TNBC pathophysiology and could be a potential therapeutic target for TNBC.