Recessive Dystrophic epidermolysis bullosa (RDEB) is a skin genetic disease caused by mutations in COL7A1 (coding type VII collagen). Type VII collagen is essential to maintain the fully functional dermal-epidermal junction, and the loss of type VII collagen results in detachment of epidermis. RDEB patients, without functional type VII collagen, suffer from the repetitive blistering and have high risk of early-onset aggressive squamous cell carcinoma. Currently, no fundamental treatment is available for RDEB. In this study, we aimed to develop therapeutics for RDEB using HMGB1 peptide, which we have previously identified as an endogenous SOS signal that can activates bone marrow stem/progenitor cells. Systemic injection of HMGB1 peptide in DEB model mice ameliorated mitten deformity and stenosis, and significantly extended the survival. This HMGB1 peptide treatment model provides a unique opportunity to investigate on how the DEB skin can be reconstructed. Thus, we comprehensively characterized the transcriptome (by RNA-seq) and epigenome (by ATAC-seq) of the skin from the HMGB1 peptide treated DEB model mice with single-cell resolution. Decrease in the number of immune cells upon the HMGB1 treatment was identified by both single cell RNA- and ATAC-seq analyses, indicating the treatment suppressed the inflammation. Furthermore, single cell ATAC-seq analysis depicted a sign of massive reprogramming in the transcription factor usage of the basal layer keratinocytes (contain stem/progenitor cells) in the HMGB1 peptide treated DEB model mice. These singe-cell level analyses suggested that the HMGB1 peptide can induce skin reconstruction by reprogramming the transcriptional program of skin stem/progenitor cells, and assured that the HMGB1 peptide serves as a promising option for the RDEB treatment.

Dystrophic epidermolysis bullosa (DEB) is a one of the most severe form of EB caused by mutations in COL7A1 (coding type VII collagen). DEB patients, without functional type VII collagen, suffer from the repetitive blistering and have high risk of early-onset aggressive squamous cell carcinoma. Recently, we found that a fragment of HMGB1 activates an endogenous tissue regeneration mechanism and ameliorates the DEB related manifestations in a DEB model mouse. This HMGB1 treatment model in the DEB mouse serves as a unique opportunity to investigate on how the skin can lose the integrity by loss of a single protein and how the damaged skin can be reconstructed. Here, we comprehensively characterized the transcriptome and epigenome of the HMGB1 treated DEB model mouse at a single cell level. We performed single cell RNA-seq and single cell ATAC-seq analyses on the skin. Both single cell RNA-seq and ATAC-seq analyses showed a decrease of immune cells by the HMGB1 treatment, indicating the treatment suppressed the inflammation. In addition, single cell RNA-seq analysis detected an increase of keratin 1/keratin 10 positive keratinocytes in the treated mice, suggesting a functional reconstruction of the DEB skin. Furthermore, single cell ATAC-seq analysis detected an increase of p63 active keratinocytes, providing another evidence for the skin reconstruction. These singe cell level data suggested that the HMGB1 treatment induced a skin reconstruction by reactivating skin stem/progenitor cells, and also serve as a foundation to design more effective DEB treatments.

Dystrophic epidermolysis bullosa (DEB) is an intractable skin genetic disease caused by mutations in the type VII collagen gene (COL7A1), which is a component of the dermal-epidermal junction. Previously, we have found that the damaged EB skin releases high mobility group box 1 (HMGB1), a transcriptional regulator known to localize in the nucleus, to mobilize bone marrow platelet-derived growth factor receptor alpha (Pdgfrα) positive cells into the damaged tissues. Here, we investigated whether the HMGB1 treatment could serve as a promising therapy for DEB. Systemic injection of HMGB1 significantly improved survival in the DEB model mice, while the untreated DEB model mice died at about 20 weeks after birth due to severe malnutrition. To understand the mechanisms of how HMGB1 ameliorates the clinical manifestations of DEB model mouse, we performed whole transcriptome analysis on the Pdgfrα positive cells at bulk and single-cell levels, and found that HMGB1 induced the anti-cell death pathway. Consistent with this anti-cell death gene profile, the number of the bone marrow Pdgfrα positive cells was maintained at physiological level in the HMGB1 treated DEB model mice, while the number of the bone marrow Pdgfrα positive cells significantly decreased without the HMGB1 treatment. Thus, these results indicated that the activation of tissue regeneration, possibly by maintaining the number of the bone marrow Pdgfrα positive cells during the course of disease, is a promising therapy for diseases of the cutaneous, and possibly non-cutaneous tissues with severe intractable damages, such as DEB.

Psoriasis is an autoimmune skin disorder characterized by severe inflammation and hyperproliferation of epidermal cells in the skin. Th17, IL-17 producing helper T, cells are known to play pivotal roles in the psoriasis pathogenesis. Although therapies targeting Th17 cells are being developed, effective treatment for psoriasis has not established. Previously, we have demonstrated that damaged tissues release high mobility group box protein 1(HMGB1) to promote tissue regeneration by mobilizing bone marrow mesenchymal stem cells (BM-MSCs). The BM-MSCs mobilized into the damaged tissues support efficient tissue regeneration by suppressing inflammation and by differentiating into multiple cell types. We have further identified the domain of HMGB1 which is essential and sufficient for activating BM-MSCs and showed that the HMGB1 domain peptide served as a promising drug for dystrophic epidermolysis bullosa (DEB), a skin genetic disorder characterized by severe blistering at skin. Because the HMGB1 therapy promotes tissue regeneration without targeting any specific molecules, this strategy can be potentially effective for a broad range of diseases with massive inflammation. Here, we tested whether the HMGB1 peptide treatment is effective for psoriasis using imiquimod (IMQ)-induced psoriasis-like dermatitis as a model. The systemic injection of HMGB1 peptide ameliorated the inflammatory manifestations of psoriasis. Consistent with these anti-inflammation activities, HMGB1 peptide decreased the gene expression of the inflammatory cytokines (IL-17A/F, IL-6, and IL-1β), suggesting that the HMGB1 peptide treatment could suppress activated Th17 cells. These data provide a foundation for the development a novel psoriasis therapy using a single defined endogenous molecule, HMGB1.

Hemagglutinating virus of Japan envelope (HVJ-E) derived from inactivated replication-defective Sendai virus possess the various antitumor activities. HVJ-E enhances multiple antitumor immunities such as activation of dendritic cells, induction of natural killer cells and CTL, and suppression of regulatory T cells, and it induces direct tumor-killing by the induction of cell death through the RIG-I/MAVS pathway by direct administration into the tumor. We performed the phase I dose escalation safety/tolerability and preliminary efficacy study of intra-tumoral and subcutaneous administration of HVJ-E in patients suffering from chemotherapy-resistant malignant pleural mesothelioma. We performed the dose upward titration clinical study for checking the safety, drug tolerance, and preliminary efficacy of the intra-tumoral and subsequent subcutaneous administration of HVJ-E. We administrated HVJ-E to the patients 4 times per 2 weeks (the first was intra-tumoral, and residual 3 times were subcutaneous injection), and then washed out the drug from the body for 2 weeks. This cycle was repeated 2 times. We observed the patients for 8 weeks, and evaluated them by CTCAE, modified RECIST, and PERCIST. Three patients were enrolled as a low-dose group, and three patients were enrolled as a high-dose group. There was no discontinuation of the administration due to the severe adverse events. Results; The mean disease duration from confirmed diagnosis to this trial was 2.27 years (0.6-4.5 years). We defined the primary endpoint as an assessment of dose limiting toxicity related with HVJ-E. Neither serious adverse events (SAE) nor DLT were observed during the observation period. The following symptoms were observed. Fever (83.3%), and the local symptoms at injection site, for example, rubor, swelling, or induration (100%) were observed, but the local relapse of mesothelioma at the injection site was not observed. It was confirmed that the intra-tumoral and subcutaneous administration of HVJ-E was safe for chemotherapy-resistant pleural mesothelioma patients, because these AEs were transient and slight. The efficacy as a secondary endpoint was evaluated with modified RECIST, and PERCIST. DCR of low dose level cohort was 0% (0/3), because of PD, meanwhile, high dose level cohort indicated 100% (3/3). Consequently, the DCR of all cases who had treated with HVJ-E was 50% (3/6) by mRECIST, meanwhile, the DCR evaluated by PERCIST was 100%. It was suggested that HVJ-E was useful for disease control of advanced pleural mesothelioma patients without severe adverse events. Now we do the next step trial for malignant pleural mesothelioma and melanoma with high dose of HVJ-E.

High mobility group box 1 (HMGB1) mainly localizes in the nucleus and regulates transcription by modulating chromatin structure. In a necrotic condition, HMGB1 is released into the extracellular milieu and plays pleiotropic roles in tissue regeneration and immunological processes. Previously, we have reported that the released HMGB1 mobilizes platelet-derived growth factor receptor alpha (PDGFRα) positive bone marrow cells into the damaged tissues using SDF-1α/CXCR4 axis. We have also shown that the HMGB1-induced PDGFRα positive cells can differentiate into both fibroblasts and keratinocytes, and provide type VII collagen (Col7) at the cutaneous basement membrane zone. Based on these results, we hypothesized that HMGB1 can serve as a potential drug for moderate form of dystrophic epidermolysis bullosa (DEB), which possess amino acid substitution mutations at least in one allele of Col7a1 gene to produce less-functional anchoring fibrils and induce dermal-epidermal separation. To test this, we systemically administered HMGB1 to Col7-hypomorphic mice. The treatment with HMGB1 significantly extended the survival with restoring the expression of Col7 at the basement membrane of the skin and the mucosa, while most of the untreated mice died within 20 weeks due to severe malnutrition. Correlated with the recovery of Col7 expression, the DEB symptoms were ameliorated. Collectively, these data position HMGB1 as a promising drug at least for moderate forms of DEB. In addition, our study provides a proof-of-concept for a drug which mobilizes multipotent cells from bone marrow, as a promising therapy for diseases of the cutaneous, and possibly non-cutaneous tissues with severe intractable damages, such as DEB.

Tissue regeneration requires efficient and coordinated replenishment of lost cells during tissue damage. In case of the limited sized tissue damage, cells can be replenished locally from neighboring healthy tissues. However, when the damage is massive, an additional mechanism to replenish cells are required for efficient tissue regeneration. Previously, we have shown that bone marrow cells function as the additional source to replenish cells to massively damaged tissues in mouse epidermolysis bullosa model, in which the epidermal-dermal junction at skin is massively disrupted due to a mutation in type VII collagen gene (Col7a1). Furthermore, we have clarified that the bone marrow cells are recruited by extracellular high mobility group box 1 (HMGB1), express platelet-derived growth factor receptor alpha (Pdgfrα) as a marker and can differentiate into multiple cell types, including epithelial cells expressing keratin 5. These data suggest that, when the damage is massive, cells are likely supplied via the circulation system for efficient tissue regeneration. However, these bone marrow cells involved in tissue regeneration have not successfully detected while the cells are circulating and analyzed its cell identity. Here, we used HMGB1 as a tool to mobilize bone marrow cells into the circulation and analyzed their characters. Systemic injection of HMGB1 significantly increased colony forming cells in the circulation and those HMGB1 mobilized cells showed higher proliferation capacity. In addition, we found that mesenchymal stem/stromal cells (MSCs), defined by its multipotency, are enriched in the circulation upon the HMGB1 injection. Our data clearly indicate that MSCs can be mobilized into the circulation by HMGB1 and suggest that tissue regeneration can be enhanced by a single defined factor. We will test the possibility of the use of HMGB1 as therapeutics for diseases with massive tissue damages, including epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is one of the most severe genetic blistering skin diseases caused by genetic dysfunction of type VII collagen (Col7), which anchor cutaneous basement membrane to the underlying dermis. Previously, we reported that necrotic epithelia of RDEB mouse skin released high mobility group box 1 (HMGB1), which then mobilized bone marrow mesenchymal stromal cells (MSCs) into the circulation, resulting in accumulation of MSCs in the RDEB mouse skin and promoting skin regeneration by anti-inflammatory and multi-differentiating activities of MSCs. We also demonstrated that transplanted bone marrow-derived MSCs could provide Col7 at the cutaneous basement membrane zone in the RDEB mouse skin. Under these backgrounds, we performed clinical trial of allogeneic MSC transplantation in three adult RDEB patients with chronic intractable ulcers continuing more than 5 years. Practically, 0.5 million of MSCs cultured from sex-mismatched healthy family donor bone marrow were subcutaneously inoculated at every 2cm distance around the chronic ulcer, and evaluated the ulcer area, expression of Col7 mRNA/ protein, formation of anchoring fibrils, and engraftment of the transplanted MSCs before and one year after the transplantation. All three cases showed almost complete healing of the intractable ulcers after one year. Among the three patients, one patient clearly demonstrated increase of Col7 protein and anchoring fibrils at the basement membrane, and two patients showed less but some engraftment of the allogeneic MSCs even after 1 year. These data clearly demonstrated that subcutaneous transplantation of allogeneic MSCs would be a therapeutic option for years-lasting intractable ulcers in severe generalized RDEB patients.

To enter the realm of human gene therapy, a novel drug delivery system is required for efficient delivery of small molecules with high safety for clinical usage. We have developed a unique vector "HVJ-E (hemagglutinating virus of Japan-envelope)" that can rapidly transfer plasmid DNA, oligonucleotide, and protein into cells by cell-fusion. In this study, we associated HVJ-E with magnetic nanoparticles, which can potentially enhance its transfection efficiency in the presence of a magnetic force. Magnetic nanoparticles, such as maghemite, with an average size of 29 nm, can be regulated by a magnetic force and basically consist of oxidized Fe which is commonly used as a supplement for the treatment of anemia. A mixture of magnetite particles with protamine sulfate, which gives a cationic surface charge on the maghemite particles, significantly enhanced the transfection efficiency in an in vitro cell culture system based on HVJ-E technology, resulting in a reduction in the required titer of HVJ. Addition of magnetic nanoparticles would enhance the association of HVJ-E with the cell membrane with a magnetic force. However, maghemite particles surface-coated with heparin, but not protamine sulfate, enhanced the transfection efficiency in the analysis of direct injection into the mouse liver in an in vivo model. The size and surface chemistry of magnetic particles could be tailored accordingly to meet specific demands of physical and biological characteristics. Overall, magnetic nanoparticles with different surface modifications can enhance HVJ-E-based gene transfer by modification of the size or charge, which could potentially help to overcome fundamental limitations to gene therapy in vivo.

This paper aims to investigate the anti-tumor mechanism of inactivated Sendai virus (Hemagglutinating virus of Japan envelope, HVJ-E) for murine melanoma (B16F10).The murine dendritic cells (DCs) were treated with HVJ-E, and then the cytokines secreted from DCs and costimulation-related molecules on DCs were measured. Meanwhile, the expression of β-catenin in HVJ-E treated murine melanoma cells was detected. In addition, HVJ-E was intratumorally injected into the melanoma on C57BL/6 mice, and the immune cells, CTL response and tumor volume were analyzed.HVJ-E injected into B16F10 melanoma obviously inhibited the growth of the tumor and prolonged the survival time of the tumor-bearing mice. Profiles of cytokines secreted by dendritic cells (DCs) after HVJ-E stimulation showed that the number of cytokines released was significantly higher than that elicited by PBS (1P<0.05). The co-stimulation-related molecules on DCs were comparable to those stimulated by LPS. Immunohistochemical examinations demonstrated the repression of β-catenin in B16F10 melanoma cells after HVJ-E treatment. Meanwhile, real-time reverse transcription PCR revealed that HVJ-E induced a remarkable infiltration of CD11c positive cells, chemokine ligand 10 (CXCL10) molecules, interleukin-2 (IL-2) molecule, CD4+ and CD8+ T cells into HVJ-E injected tumors. Furthermore, the mRNA expression level of β-catenin in the HVJ-E injected tumors was also down-regulated. In addition, B16F10-specific CTLs were induced significantly after HVJ-E was injected into the tumor-bearing mice.This is the first report to show the effective inhibition of melanoma tumors by HVJ-E alone and the mechanism through which it induces antitumor immune responses and regulates important signal pathways for melanoma invasion. Therefore, HVJ-E shows its prospect as a novel therapeutic for melanoma therapy.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and –liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse model compared to the BCG. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality. The BCG prime and HSP65+IL-12/HVJ vaccine (boost) by the prime-boost method showed a synergistic prophylactic effect in the monkey. Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys.HVJ-Envelope/HSP65 DNA+IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the ESR, and augmented the immuneresponses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials.