Induction of potent antitumor immunity by intradermal DNA injection using a novel needle-free pyro-drive jet injector.

The current success of mRNA vaccines against COVID-19 has highlighted the effectiveness of mRNA and DNA vaccinations. Recently, we demonstrated that a novel needle-free pyro-drive jet injector (PJI) effectively delivers plasmid DNA into the skin, resulting in protein expression higher than that achieved with a needle syringe. Here, we used ovalbumin (OVA) as a model antigen to investigate the potential of the PJI for vaccination against cancers. Intradermal injection of OVA-expression plasmid DNA into mice using the PJI, but not a needle syringe, rapidly and greatly augmented OVA-specific CD8+ T-cell expansion in lymph node cells. Increased mRNA expression of both interferon-γ and interleukin-4 and an enhanced proliferative response of OVA-specific CD8+ T cells, with fewer CD4+ T cells, were also observed. OVA-specific in vivo killing of the target cells and OVA-specific antibody production of both the IgG2a and IgG1 antibody subclasses were greatly augmented. Intradermal injection of OVA-expression plasmid DNA using the PJI showed stronger prophylactic and therapeutic effects against the progression of transplantable OVA-expressing E.G7-OVA tumor cells. Even compared with the most frequently used adjuvants, complete Freund's adjuvant and aluminum hydroxide with OVA protein, intradermal injection of OVA-expression plasmid DNA using the PJI showed a stronger CTL-dependent prophylactic effect. These results suggest that the novel needle-free PJI is a promising tool for DNA vaccination, inducing both a prophylactic and a therapeutic effect against cancers, because of prompt and strong generation of OVA-specific CTLs and subsequently enhanced production of both the IgG2a and IgG1 antibody subclasses.

Mechanism of jet injector-induced plasmid DNA uptake: Contribution of shear stress and endocytosis.

The administration of plasmid DNA (pDNA) using a pyro-drive jet injector allows gene expression in cells of the treated tissue; however, the detailed plasmid uptake process remains to be determined. A recent theory suggests that shear stress enhances the endocytosis pathway and pDNA internalization. Here, we investigated the process of pDNA uptake in the context of a pyro-drive jet injector-based administration as a way to optimize gene transfer efficiency via the increase in DNA uptake. The gene expression was significantly improved when the shear stress caused by the jet was generated where the pDNA was retained. Contrarily, heparin, an inhibitor of the spontaneous uptake of injected DNA, inhibited the gene expression in jet injection. In addition, treatment with typical endocytosis inhibitors (chlorpromazine, methyl-ß-cyclodextrin, dimethyl amiloride, rottlerin, and NSC23766) also reduced plasmid expression efficiency in the context of jet injection; conversely, endosome escape in the context of chloroquine treatment increased the expression efficiency. Altogether, our results not only clarify the mechanism of pDNA uptake in the context of jet injection but also highlight the key role of endosomes on the intracellular trafficking of pDNA. Importantly, such findings may impact other studies on gene transfer and endocytosis and boost further efforts to improve the efficiency and safety of jet injection in the context of both basic and translational applications.

Title: Gene transfer by pyro-drive jet injector is a novel therapeutic approach for muscle diseases.

The angiogenic gene therapy is an attractive approach for the treatment of ischemic muscle diseases, including peripheral arterial disease and ischemic heart diseases. Although a variety of gene transfer methods have been developed, the efficiency of gene transfer is still limited. We have been developing the needleless high-energy bioinjector device, Pyro-drive Jet Injector (PJI), based on pyrotechnics using a combination of ignition powder and gunpowder, however, the utility of PJI in gene transfer into muscle tissues remains unclear. pcDNA3.1 plasmid containing Flag was injected to the thigh muscles of C57BL/6J mice using PJI or needle, as a control. Histological analysis demonstrated that the protein expression of Flag was observed in a wider range in PJI group than in needle group. To assess the validity of PJI for gene therapy, pcDNA3.1-human fibroblast growth factor 2 (FGF2), which has angiogenic activity and tissue protective properties, was injected into the ischemic thigh muscles with PJI or needle. ELISA assay revealed that the protein expression of FGF2 was increased in the thigh muscle tissues by PJI-mediated gene delivery. Significantly, histological analyses revealed that muscle fiber cross-sectional area and the number of endothelial marker CD31 (+) cells was increased in ischemic hind-limb tissues of the PJI-FGF2 group but not in those of needle-FGF2 group. To expand the applicability of the PJI-mediated gene transfer, pcDNA3.1-venus plasmid was injected into murine hearts with PJI or needle. PJI method was successful in gene transfer into murine hearts, especially into cardiomyocytes, with high efficiency when compared to needle method. Collectively, the non-needle, non-liposomal and non-viral gene transfer by PJI could be a novel therapeutic approach for muscle diseases.

Potent Intradermal Gene Expression of Naked Plasmid DNA in Pig Skin Following Pyro-drive Jet Injection.

Intradermal administration of naked DNA with a conventional needle syringe is a simple and inexpensive method to expose an encoded antigen to the dermal immune system. We aimed to enhance intradermal gene expression with a pyro-drive jet injector using pig skin, which is similar in structure and biomechanical properties to human skin. When Cy3-labeled plasmid (pCy3) was applied to pig skin with the jet injector, pCy3 was distributed preferentially in the intradermal tissue. Precise localization analysis revealed that pCy3 was also detected in the intracellular nucleus, and the frequency was substantially higher with the jet injector than with a needle syringe. When a luciferase expression plasmid (pLuc) was injected transdermally, the luciferase activity was 380-fold higher with the jet injector than with a needle syringe. Furthermore, immunohistochemistry analysis showed that the epidermis was positive for luciferase protein expression. These data indicate that the jet injector facilitates stable intradermal administration, resulting in more efficient gene expression compared to that with conventional syringe methods. Thus, intradermal administration of an antigen-expression plasmid with the pyro-drive jet injector may provide a clinically viable method for future gene therapy.

Development of Pyro-Drive Jet Injector With Controllable Jet Pressure.

Various jet injectors have been developed and used for the effective and efficient administration of drugs. Jet injections overcome the limitations of other drug delivery methods, such as ablation, iontophoresis, electroporation, sonophoresis, and microneedles, because jet injection is not limited by the diffusion rates of different drugs. However, controlling the jet pressure during drug delivery is difficult with most conventional jet injectors. Efficacy evaluation of such devices on laboratory animals is strongly required before initiating human clinical trials, but minimal research has been performed for the device developments. Therefore, we developed jet injector devices based on pyrotechnics using 2 types of explosives with different burning rates; we call these pyro-drive jet injectors. The liquid jet pressure profile suggests that the penetration depth and injection volume for soft materials and skin tissue are controllable. Here, we propose the pyro-drive jet injectors as another candidate well-controlled jet injector for laboratory animals in drug discovery testing as well as human clinical use.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia.

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3' UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

Comparison of chronic renal failure rats and modification of the preparation protocol as a hyperphosphataemia model.

BACKGROUND: Several animal models with chronic renal failure have been established and used for demonstrating complications including hyperphosphataemia. Although long-time feeding is required to cause hyperphosphataemia in animals, a few modifications have been reported to provide more useful models for research. METHODS: Three separate experiments were carried out in the present study. First, characteristics of commonly used subnephrectomized (5/6Nx) rats and rats fed an adenine diet (0.75% adenine in normal diet) were compared as hyperphosphataemia models. Next, using adenine-diet rats, the inhibitory effect of sevelamer hydrochloride (Sev) on serum phosphorus elevation was examined. Third, oral adenine dosing for induction of hyperphosphataemia and validation as a model using Sev were examined. RESULTS: Serum phosphorus in 5/6Nx rats became elevated in 8-17 weeks, but the levels and time points of elevation differed among animals. In adenine-fed rats, the elevation was more clearly demonstrated with less diversity at 4 weeks. The data revealed a potential shorter model preparation period and the importance of controlling feeding amounts. Oral adenine dosing induced hyperphosphataemia by 12 days, and Sev treatment was inhibitory. After a maintenance period of over a month (no treatments), Sev-treated rats showed hyperphosphataemia as did oral adenine-dosed control rats. The serum phosphorus levels significantly decreased on further Sev treatment. CONCLUSION: Oral dosing with adenine made the model preparation period definitely shorter, and its usefulness as a hyperphosphataemia model was revealed using Sev.

Exclusive association and simultaneous appearance of congophilic plaques and AT8-positive dystrophic neurites in Tg2576 mice suggest a mechanism of senile plaque formation and progression of neuritic dystrophy in Alzheimer's disease.

Progression of neuritic dystrophy is a histological hallmark of Alzheimer's disease (AD) in addition to amyloid deposition and neurofibrillary tangle formation. Dystrophic neurites (DNs) are abnormal neurites, and are closely associated with amyloid deposits. To clarify the process of DN formation, we immunohistochemically investigated phosphorylated tau (AT8 and Ser396)-positive DNs and plaques in Tg2576 mice overexpressing human beta-amyloid precursor protein (APP) with the Swedish type mutation (K670N/M671L). AT8-positive DNs were exclusively associated with the Congo red-positive plaques examined, and all Abeta(1-40)-positive plaques appeared to be associated with AT8-positive DNs, whereas there were no AT8-positive DNs with Abeta(1-42)-positive/Abeta(1-40)-negative plaques. Since we have previously shown that Abeta(1-42)-positive plaque precede Abeta(1-40) deposition, the appearance of congophilic structures is also late. Quantitative analyses were performed on AT8-positive DNs that were associated with congophilic plaques in the cerebral cortex and hippocampus (more than 1,000 plaques). The number of congophilic plaques increased dramatically with age. The area of DNs in the cerebral cortex and hippocampus increased 120- and 60-fold from 11-13 to 20.5 months of age, respectively. Interestingly, the mean ratio of DN area to congophilic plaque area in every plaque was unchanged, approximately 10%, through the ages examined. The mean plaque size was stable with age in both the cortex and hippocampus. These data suggest that the formation of AT8-positive DNs is simultaneous with Congo red-positive plaque development, and that the event may be closely related in the pathological progression of AD.

Novel insertional mutation in the bone morphogenetic protein receptor type II associated with sporadic primary pulmonary hypertension.

Primary pulmonary hypertension (PPH), which results from occlusion of small pulmonary arteries, is a devastating condition. Mutations of the bone morphogenetic protein receptor type II gene (BMPR2), a component of the transforming growth factor- beta (TGF-beta) family, which plays a key role in cell growth, have recently been identified as causing familial and sporadic PPH. The first case of BMPR2 mutation found in Japan is reported here in a 19-year-old woman with a clinical diagnosis of PPH and no identifiable family history of pulmonary hypertension. Direct sequencing of the entire coding region and intron/exon boundaries of BMPR2 revealed a frameshift mutation predicted to alter the cell signaling response to specific ligands. A molecular classification of PPH, based upon the presence or absence of BMPR2 mutations, might have important implications for patient management and screening of relatives.

Amount of bleeding and hematoma size in the collagenase-induced intracerebral hemorrhage rat model.

The aggravated risk on intracerebral hemorrhage (ICH) with drugs used for stroke patients should be estimated carefully. We therefore established sensitive quantification methods and provided a rat ICH model for detection of ICH deterioration. In ICH intrastriatally induced by 0.014-unit, 0.070-unit, and 0.350-unit collagenase, the amount of bleeding was measured using a hemoglobin assay developed in the present study and was compared with the morphologically determined hematoma volume. The blood amounts and hematoma volumes were significantly correlated, and the hematoma induced by 0.014-unit collagenase was adequate to detect ICH deterioration. In ICH induction using 0.014-unit collagenase, heparin enhanced the hematoma volume 3.4-fold over that seen in control ICH animals and the bleeding 7.6-fold. Data suggest that this sensitive hemoglobin assay is useful for ICH detection, and that a model with a small ICH induced with a low-dose collagenase should be used for evaluation of drugs that may affect ICH.

Effect of AMPA receptor antagonist YM872 on cerebral hematoma size and neurological recovery in the intracerebral hemorrhage rat model.

[2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]-acetic acid monohydrate (YM872 or zonampanel), an AMPA receptor antagonist, is in clinical development for acute ischemic cerebral infarction. Stroke patients are prone to have subsequent intracerebral hemorrhages. In order to predict potential adverse effects, YM872 was tested in a rat model with collagenase-induced intracerebral hemorrhage. The morphologically determined hematoma volumes after 24 h were compared between animal groups intravenously infused with 3600 U/kg/h heparin for 30 min, or with 20 or 40 mg/kg/h of YM872, or placebo for 4 h. Heparin enlarged hematoma volume, but neither dose of YM872 affected hematoma size. In a separate study, neurological deficits were scored at various days after intracerebral hemorrhage induction in animals with intravenous infusion for 24 h of 10 or 20 mg/kg/h YM872, or saline. The YM872 groups scored significantly better than the saline group at 14 days. These data suggest that YM872 does not exacerbate intracerebral hemorrhage and might accelerate recovery.