Cellular calcium oscillations in droplets with different chemical concentrations supplied by droplet-array sandwiching technology

DAST技術の独自展開によりバイオケミカル分野への幅広い応用を期待 --微量液滴間の接触によりヒスタミン供給量を調整し液滴内の細胞カルシウム振動の変化を初実証--. 京都大学プレスリリース. 2022-08-10.Digital microfluidics using droplets on a chip, such as droplet-array sandwiching technology, provide efficient tools for biochemistry from the perspective of time and sample consumption. Droplet-array sandwiching technology uses the fusion and separation of droplets on upper and lower substrates for high-throughput screening. In our previous work, we developed independent control of individual droplets for this technology using electrowetting-on-dielectric to control the droplet height and allow different chemical concentrations in droplets on the same chip. In this study, we explored the applicability of droplet-array sandwiching technology to cell-based analysis by observing cellular calcium oscillations in HeLa cells in droplets with different histamine concentrations. Histamine concentrations could be controlled by controlling contact time between upper and lower droplets. We found that calcium oscillations intensified with higher histamine concentrations prepared by controlling contact time. These results suggest that droplet-array sandwiching technology can be used for cell-based analysis, where the chemical concentration for cellular stimulation needs to be controlled

Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase.

PhiC31 integrase-based vectors can integrate therapeutic genes selectively into attP or pseudo-attP sites in genomes, but considerable numbers of pseudo-attP sites in human genomes exist inside endogenous gene-coding regions. To avoid endogenous gene disruptions, we aimed to enhance the integration site-specificity of the phiC31 integrase-based vector using a sequence-specific DNA-binding protein containing Gal4 and LexA DNA-binding motifs. The dual DNA-binding protein was designed to tether the UAS-containing donor vector to the target sequence, the LexA operator, and restrict integration to sites close to the LexA operator. To analyze the site-specificity in chromosomal integration, a human cell line having LexA operators on the genome was established, and the cell line was transfected with donor vectors expressing the DNA-binding protein and the phiC31 integrase expression vector (helper vector). Quantitative PCR indicated that integration around the LexA operator was 26-fold higher with the UAS-containing donor vector than with the control. Sequence analysis confirmed that the integration occurred around the LexA operator. The dual DNA-binding protein-based targeted integration strategy developed herein would allow safer and more reliable genetic manipulations for various applications, including gene and cell therapies

piggyBac Transposon-mediated Long-term Gene Expression in Mice

Transposons are promising systems for somatic gene integration because they can not only integrate exogenous genes efficiently, but also be delivered to a variety of organs using a range of transfection methods. piggyBac (PB) transposon has a high transposability in mammalian cells in vitro, and has been used for genetic and preclinical studies. However, the transposability of PB in mammalian somatic cells in vivo has not been demonstrated yet. Here, we demonstrated PB-mediated sustained gene expression in adult mice. We constructed PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase (Gluc)] genes. Mice were transfected by injection of these pDNAs using a hydrodynamics-based procedure, and the conditions for high-level sustained gene expression were examined. Consequently, gene expressions were sustained over 2 months. Our results suggest that PB is useful for organ-selective somatic integration and sustained gene expression in mammals, and will contribute to basic genetic studies and gene therapies

Sialyl LewisX mimic-decorated liposomes for anti-angiogenic everolimus delivery to E-selectin expressing endothelial cells

In this study, we developed novel E-selectin-targeting liposomes, i.e., 3′-(1-carboxy)ethyl sialyl LewisX (3′-CE sLeX) mimic liposomes, for targeted delivery of everolimus (EVE) in anti-angiogenic therapy. We investigated the uptake and efficacy of these E-selectin targeting liposomes in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs). The uptake of EVE in 3′-CE sLeX mimic liposomes increased steadily and almost caught up with the uptake of plain EVE at 3 h, which was higher than that in PEGylated liposomes (PEG-liposomes). Inhibition of uptake by anti-E-selectin antibody suggested involvement of E-selectin-mediated endocytotic processes. Migration in cells treated with EVE/3′-CE sLeX mimic liposomes was suppressed by more than half when compared to the control. This treatment was also seen to significantly inhibit the formation of capillary tubes and networks. In addition, Thr389 phosphorylation of pS6 kinase, as a marker of mTOR activity, was remarkably suppressed to less than endogenous levels by EVE/3′-CE sLeX mimic liposomes. In conclusion, the present study demonstrated that EVE/3′-CE sLeX mimic liposomes were intracellularly taken up by E-selectin and prompted anti-angiogenic effects of EVE involved in the mTOR signaling pathway. However, moderate retention of EVE in the liposomes might limit the targeting ability of 3′-CE sLeX mimic liposomes

Antitumor effect of nuclear factor-κB decoy transfer by mannose-modified bubble lipoplex into macrophages in mouse malignant ascites

Patients with malignant ascites (MAs) display several symptoms, such as dyspnea, nausea, pain, and abdominal tenderness, resulting in a significant reduction in their quality of life. Tumor-associated macrophages (TAMs) play a crucial role in MA progression. Because TAMs have a tumor-promoting M2 phenotype, conversion of the M2 phenotypic function of TAMs would be promising for MA treatment. Nuclear factor-κB (NF-κB) is a master regulator of macrophage polarization. Here, we developed targeted transfer of a NF-κB decoy into TAMs by ultrasound (US)-responsive, mannose-modified liposome/NF-κB decoy complexes (Man-PEG bubble lipoplexes) in a mouse peritoneal dissemination model of Ehrlich ascites carcinoma. In addition, we investigated the effects of NF-κB decoy transfection into TAMs on MA progression and mouse survival rates. Intraperitoneal injection of Man-PEG bubble lipoplexes and US exposure transferred the NF-κB decoy into TAMs effectively. When the NF-κB decoy was delivered into TAMs by this method in the mouse peritoneal dissemination model, mRNA expression of the Th2 cytokine interleukin (IL)-10 in TAMs was decreased significantly. In contrast, mRNA levels of Th1 cytokines (IL-12, tumor necrosis factor-α, and IL-6) were increased significantly. Moreover, the expression level of vascular endothelial growth factor in ascites was suppressed significantly, and peritoneal angiogenesis showed a reduction. Furthermore, NF-κB decoy transfer into TAMs significantly decreased the ascitic volume and number of Ehrlich ascites carcinoma cells in ascites, and prolonged mouse survival. In conclusion, we transferred a NF-κB decoy efficiently by Man-PEG bubble lipoplexes with US exposure into TAMs, which may be a novel approach for MA treatment. We demonstrated that the combinatorial use of mannose-modified bubble lipoplexes with ultrasound exposure achieved the efficient delivery of NF-κB decoy oligonucleotides into tumor-associated macrophages (TAM) in a mouse peritoneal dissemination model of Ehrlich ascites carcinoma. Using this method, NF-κB decoy transfer into TAM inhibited the malignant ascites progression significantly that may be the phenotypic conversion of TAM from M2 toward M1

Gamma-polyglutamic acid-coated vectors for effective and safe gene therapy.

In the present study, we developed some novel gene delivery vectors, coated cationic complexes with gamma-polyglutamic acid (gamma-PGA) for effective and safe gene therapy. Cationic complexes were constructed with pDNA and cationic vectors, such as poly-L-arginine hydrochloride (PLA), poly-L-lysine hydrobromide (PLL), N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA)-cholesterol (Chol) liposomes, and DOTMA-dioleylphosphatidylethanolamine (DOPE) liposomes. The cationic complexes showed high gene expression with strong cytotoxicity in melanoma B16-F10 cells. The cationic complexes were also strongly toxic to erythrocytes. On the other hand, the gamma-PGA was able to coat all cationic complexes and form stable nano-sized particles with negative charges. These gamma-PGA-coated complexes had high gene expression without cytotoxicity and toxicities to the erythrocytes. In in vivo transfection experiments, polyplexes showed high transfection efficiency over 10(5) RLU/g in the lung tissue after intravenous injection, although gamma-PGA-coated polyplexes showed a high value in the spleen. High transfection efficiency in lipoplexes and gamma-PGA-coated lipoplexes was observed in the spleen and lung. Thus, gamma-PGA-coated vectors are useful for clinical gene therapy

Development of anionic bubble lipopolyplexes for efficient and safe gene transfection with ultrasound exposure in mice

Anionic bubble lipopolyplexes have been developed as anionic ultrasound (US)-responsive gene delivery carriers with biocompatible compounds for efficient and safe transfection in mice. The particles of the anionic bubble lipopolyplexes were approximately 450-600 nm with an anionic surface charge. In the absence of US exposure, the bubble lipopolyplexes showed extremely low gene expression in the human vascular endothelial cell line EAhy926. The anionic bubble lipopolyplexes, however, delivered pDNA into cells without endocytosis and showed markedly high gene expression following US exposure. The anionic bubble lipopolyplexes showed little cytotoxicity in EAhy926 cells and little aggregation with erythrocytes. Following intravenous administration into mice, the anionic bubble lipopolyplexes showed high levels of gene expression in the liver, kidney, and spleen only after US exposure to the abdominal area. The level of gene expression in liver non-parenchymal cells was significantly higher than that in parenchymal cells. In addition, the anionic bubble lipopolyplexes did not show any severe hepatic toxicity and did not enhance the production of proinflammatory cytokines. Overall, we have succeeded in preparing anionic bubble lipopolyplexes for efficient and safe transfection with US exposure in mice

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Evaluation of osteoclastogenesis via NFκB decoy/mannosylated cationic liposome-mediated inhibition of pro-inflammatory cytokine production from primary cultured macrophages.

To explore the effect of NFκB activation in macrophages on osteoclastogenesis of bone marrow cells for potential application as a new type of therapy for preventing bone loss