Biology subjects

Kamoshita, N.

Publications and source records attributed to Kamoshita, N..

4 recordsLinked to original sources

bioRxiv2025

Epithelial cells induce the polar migration via the MHC-I-AltR interaction to eliminate transformed cells

In epithelial tissues, adjacent normal cells recognize and eliminate transformed cells, maintaining tissue homeostasis. We have demonstrated that the interaction between MHC-I (Major Histocompatibility Complex Class I) on transformed cells and the receptor AltR (Suboptimal Alteration Recognizing Protein) on the normal epithelial cell plays a central role in triggering the elimination ability within normal cells. Moreover, previous reports have suggested that the collective movement of surrounding normal cells including "non-adjacent peripheral normal cells" which are located further out from transformed cells also contributes to the elimination of transformed cells. However, how the direct interaction mediated by MHC-I-AltR affects the behavior of these peripheral normal cells remained unclear. In this study, we aimed to analyze the relationship between the collective movement of normal cells and the elimination of transformed cells. To achieve this, we visualized the two-dimensional movement of individual epithelial cells using the PIV (Particle Image Velocimetry) analysis and quantified the behavior of transformed cells and surrounding normal cells as vectors composed of speed and direction. As a result, we observed that a subset of normal cells near the transformed cells exhibited the unidirectional migration toward the transformed cells (polar migration), which ultimately led to their elimination. Furthermore, this polar migration was suggested to be induced by the MHC-I-AltR interaction. Additional analyses suggest that MHC-I-stimulated AltR induces Ca2+ signaling between normal cells, which in turn triggers the polar migration. Based on these findings, we conclude that the core molecules involved in transformed recognition, MHC-I and AltR, not only trigger the elimination ability in adjacent normal cells but also regulate the behavior of non-adjacent peripheral normal cells, contributing to the elimination of transformed cells.

molecular biology↗

bioRxiv2023

Cure of congenital purpura fulminans via expression of engineered protein C through neonatal genome editing in mice

Protein C (PC) is a plasma anticoagulant encoded by PROC; mutation in both PROC alleles results in neonatal purpura fulminans--a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency. First, we generated an engineered activated PC to insert a self-cleaving peptide sequence between light and heavy chains. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of protein S in vitro. The adeno-associated virus (AAV) vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation factor V in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6 mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using AAV vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo. These results suggest that the expression of the engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency. One Sentence SummaryEctopic expression of an engineered protein C via genome editing cures protein C deficiency in mice.

bioengineering↗

bioRxiv2022

Efficient Gene Transduction in Pigs and Macaques with the Engineered AAV Vector AAV.GT5 for Hemophilia B Gene Therapy

Gene therapy for hemophilia using adeno-associated virus (AAV) vectors allows long-term coagulation factor expression. We examined the potential of a novel engineered liver-tropic AAV3B-based vector AAV.GT5 for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, while in vivo transduction efficacy into the liver and the increase in coagulation factor IX (FIX) antigen following intravenous injection of these vectors were similar in PXB mice (chimeric mice with a humanized liver) and macaques. The discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the original AAV3B. The intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced vector transduction into the liver and reduced vector distribution to the kidney in pigs. In macaques, the intra-hepatic artery injection of AAV.GT5 yielded a comparable increase in FIX antigen with a one-third dosage of peripheral venous administration. Two of four macaques who received AAV.GT5 intravenously did not develop neutralizing antibodies (NAbs) against AAV.GT5, while AAV-Spark100 induced serotype-specific NAbs in all four macaques. The NAb produced after the administration was relatively specific to the serotype and less responsive to the other serotype. As a result, the administration of AAV.GT5 successfully boosted FIX expression in one animal previously given AAV-Spark100. Thus, AAV.GT5 has different biodistribution and immunogenic characteristics compared with AAV-Spark100, and the intra-hepatic vascular administration may lessen the vector dose and avoid vector distribution to other organs. Key PointsO_LIThe AAV.GT5 vector has a strong transduction efficacy in human hepatocytes but has a faster clearance after systemic administration. C_LIO_LIIntra-hepatic vascular administration of the AAV.GT5 vector is an effective liver transduction method for hemophilia gene therapy. C_LI

genetics↗

bioRxiv2022

Successful Liver transduction by Re-administration of Different Adeno-Associated Virus Vector Serotypes in Mice

Intravenous administration of adeno-associated virus (AAV) vector is a promising gene therapy approach for monogenic diseases. However, re-administration of the same AAV serotype is impossible due to the induction of anti-AAV neutralizing antibodies (NAbs). Here we examined the feasibility of re-administration of AAV vectors to change the serotypes. We administered AAV3B, AAV5, or AAV8 vectors targeting the liver of C57BL/6 mice intravenously, and then assessed the emergence of NAbs and the transduction efficacy with a second administration. For all serotypes, we confirmed that re-administration with the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5; however, the NAbs elicited by AAV5 did not react with any other serotypes, resulting in success in re-administration with the other serotypes. The re-administration of AAV5 was also successful in all mice treated with AAV3B and AAV8. The effective secondary administration of AAV3B and AAV8 was observed in most mice treated with AAV8 and AAV3B, respectively. However, few mice developed NAbs cross-reactive with the other serotypes, especially the serotypes with close sequence homology. In summary, AAV vector administration induced NAbs relatively specific to the serotype administrated. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice.

molecular biology↗