p53 dry gene powder enhances anti-cancer effects of chemotherapy against malignant pleural mesothelioma.

Dry gene powder is a novel non-viral gene-delivery system, which is inhalable with high gene expression. Previously, we showed that the transfection of p16INK4a or TP53 by dry gene powder resulted in growth inhibitions of lung cancer and malignant pleural mesothelioma (MPM) in vitro and in vivo. Here, we report that dry gene powder containing p53- expression-plasmid DNA enhanced the therapeutic effects of cisplatin (CDDP) against MPM even in the presence of endogenous p53. Furthermore, our results indicated that the safe transfection with a higher plasmid DNA (pDNA) concentration suppressed MPM growth independently of chemotherapeutic agents. To develop a new therapeutic alternative for MPM patients without safety concerns over "vector doses", our in vitro data provide basic understandings for dry gene powder.

Effects of inhalable gene transfection as a novel gene therapy for non-small cell lung cancer and malignant pleural mesothelioma.

Gene therapy using vectors has attracted attention in recent years for the treatment of cancers caused by gene mutations. Besides, new treatments are imperative for lung cancer, including non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM), due to its high mortality. We developed a minimally invasive and orally inhalable tumor suppressor gene drug (SFD-p16 and SFD-p53) with non-viral vectors for lung cancer treatment by combining tumor suppressor genes with an inhalant powder that can deliver active ingredients directly to the lung. We used NSCLC (A549 and H1299) and MPM (H2052) cell lines in an air-liquid interface culture. Transfection of A549 and H2052 cells with SFD-p16 significantly increased p16 mRNA expression levels and decreased cell proliferation in both cell lines. Similar results were obtained with transfection of H1299 with the inhalable gene drug SFD-p53. In an in vivo experiment, a mouse model of lung cancer with orthotopically transplanted luciferase-expressing A549 cells was subjected to intratracheal insufflation of SFD-p16. Consequently, SFD-p16 effectively and directly affected lung cancer. This study suggests that inhalable gene drugs are effective treatments for NSCLC and MPM. We expect inhalable gene drugs to present a novel gene therapy agent for lung cancer that patients can self-administer.

Mouse-hamster chimeric prion protein (PrP) devoid of N-terminal residues 23-88 restores susceptibility to 22L prions, but not to RML prions in PrP-knockout mice.

Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp 0/0 mice, neither developed the disease nor accumulated MHM2ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice developed the disease with abundant accumulation of MHM2ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2ScΔ23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM2Δ23-88 to MHM2ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp 0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp 0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice, compared with RML- and 22L-inoculated Prnp 0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2ScΔ23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM2Δ23-88 into MHM2ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrPC into PrPSc.

Prions disturb post-Golgi trafficking of membrane proteins.

Conformational conversion of normal cellular prion protein PrP(C) into pathogenic PrP(Sc) is central to the pathogenesis of prion diseases. However, the pathogenic mechanism remains unknown. Here we show that post-Golgi vesicular trafficking is significantly delayed in prion-infected N2a cells. Accordingly, cell surface expression of membrane proteins examined, including PrP(C), insulin receptor involved in neuroprotection, and attractin, whose mutation causes prion disease-like spongiform neurodegeneration, is reduced. Instead, they accumulate in the Golgi apparatus. PrP(Sc) is detected throughout endosomal compartments, being particularly abundant in recycling endosome. We also show reduced surface expression of PrP(C) and insulin receptor in prion-infected mouse brains well before the onset of disease. These results suggest that prion infection might impair post-Golgi trafficking of membrane proteins to the cell surface in neurons via PrP(Sc) accumulated in recycling endosome, and eventually induce neuronal dysfunctions associated with prion diseases.

Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions.

Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrPΔOR)/Prnp(0/0) mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrPΔOR, or PrP(Sc)ΔOR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrP(Sc)ΔOR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrP(Sc)ΔOR, including the pre-OR residues 23-50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.

Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.

We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.