犬モデルにおける ex vivo および in vivo 遺伝子治療のための代替遺伝子導入技術の開発

Introduction: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. Methods: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. Results: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. Conclusions: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.博士（医学）・乙第1517号・令和3年12月21日© 2021, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/)

Black carbon and inorganic aerosols in Arctic snowpack

Key Points: • First ever measurements with a high‐accuracy single‐particle soot photometer of black carbon (BC) concentrations in Arctic snowpack • Topography and BC emission flux strongly influenced latitudinal variations of mass concentrations and size distributions of BC • Measured BC mass concentrations 2–25 times lower than previously reported show the importance of revalidating climate modelsBlack carbon (BC) deposited on snow lowers its albedo, potentially contributing to warming in the Arctic. Atmospheric distributions of BC and inorganic aerosols, which contribute directly and indirectly to radiative forcing, are also greatly influenced by depositions. To quantify these effects, accurate measurement of the spatial distributions of BC and ionic species representative of inorganic aerosols (ionic species hereafter) in snowpack in various regions of the Arctic is needed, but few such measurements are available. We measured mass concentrations of size-resolved BC (CMBC) and ionic species in snowpack by using a single-particle soot photometer and ion chromatography, respectively, over Finland, Alaska, Siberia, Greenland, and Spitsbergen during early spring in 2012–2016. Total BC mass deposited per unit area (DEPMBC) during snow accumulation periods was derived from CMBC and snow water equivalent (SWE). Our analyses showed that the spatial distributions of anthropogenic BC emission flux, total precipitable water, and topography strongly influenced latitudinal variations of CMBC, BC size distributions, SWE, and DEPMBC. The average size distributions of BC in Arctic snowpack shifted to smaller sizes with decreasing CMBC due to an increase in the removal efficiency of larger BC particles during transport from major sources. Our measurements of CMBC were lower by a factor of ~13 than previous measurements made with an Integrating Sphere/Integrating Sandwich spectrophotometer due mainly to interference from coexisting non-BC particles such as mineral dust. The SP2 data presented here will be useful for constraining climate models that estimate the effects of BC on the Arctic climate.Plain Language Summary Black carbon (BC) particles, commonly known as soot, are emitted from incomplete combustion of fossil fuels and biomass. They efficiently absorb solar radiation and thus heat the atmosphere. BC particles emitted at midlatitudes and in the Arctic are deposited onto snow in the Arctic, accelerating snowmelt in early spring by absorbing solar radiation. These processes contribute to warming in the Arctic. Calculations of this warming effect by using numerical models need to be validated by comparison with observed BC concentrations in snowpack. However, there are very few accurate records of concentrations of BC in snow because of technical difficulties in making these measurements. We developed a new laser-induced incandescence technique to measure BC concentrations in snowpack and applied it for the first time in six Arctic regions (Finland, Alaska, North and South Siberia, Greenland, and Spitsbergen). The BC concentrations we measured were highest in Finland and South Siberia, which are closer to large anthropogenic BC sources than the other regions, where our measured BC concentrations were much lower. On average, our BC concentrations were much lower than those previously measured by different techniques. Therefore, previous comparisons of modeled and observed BC concentrations need to be re-evaluated using the present data

チテキ ショウガイ ジ ニ オケル オンセツ チュウシュツ カダイ ト シリトリ カダイ ノ シュウトク ケイカ ニ カンスル ジレイ ケンキュウ

知的障害児を対象としたかな文字の読み書き学習では、音節抽出課題の指導が重要である。本研究では、音節抽出課題に先だって「かな文字カードにより単語を呈示した後、隠す」という手続きを利用して音節抽出の支援課題を設定し、その支援効果に関して検討することを目的とした。対象は、知的障害児2名（知的障害特別支援学校小学部4年生、語彙年齢4歳および7歳）とした。指導前において両対象児は、かな文字読みについてほぼ達成を示したが、音節抽出課題未達成を示した。7月から11月にかけての音節抽出指導の結果、対象児Ａ、Ｂ共に支援がない条件で、2、3音節単語の音節抽出課題の正答率が増加し、課題達成を確認できた。また、2から4音節単語の音節抽出が達成された段階の後に、しりとり課題の流暢な遂行が可能になった。これより音節抽出の支援課題の有効性を指摘できる

Field observations of wet snow accretion on overhead transmission lines at the Kushiro test line

Wet snow accretion on overhead lines often causes large-scale snow damage. The Kushiro test line, Japan, was constructed in 2013 for field observations of wet snow accretion in conductors and insulators as well as galloping resulting from snow accretion in overhead lines. Several cases of noticeable wet snow accretion have been observed at this site, especially in the winter of 2014. In the most notorious case, more than 2 kg/m of snow was found to be accreted on the ACSR240 single conductor, which has a diameter of 22.4 mm. In the case of single conductors without countermeasures, snow accretion developed to form a cylindrical sleeve with wire rotation. However, in some cases, the cylindrical sleeve of snow accretion also occurred in four-bundled conductors, where line spacers prevent the wire from rotating. This indicates that the accreted wet snow slides along the strands of the wire to form a cylindrical sleeve. Accordingly, the effectiveness of the snow resistance ring, which is the most commonly used anti-snow-damage device in Japan, in preventing snow accretion was confirmed. As the ring prevents the accreted snow from sliding, accretion areas tend to split, causing the snow to be shed from the conductor. More heat transfer from the air to snow and heat generated by the electric current facilitated the sliding of snow along the strand, making the snow resistance ring more effective

A novel cell-sheet technology that achieves durable factor VIII delivery in a mouse model of hemophilia A.

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3-5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A