Background: Base editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility. Methods: We generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient’s F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively. Results: Here we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG–mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor. Conclusion: A base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B.

Aim: Immunotherapies blocking the CD47‐SIRPα pathway by targeting CD47 enhance macrophage phagocytosis of neoplastic cells in mouse models. As SIRPα exhibits relatively restricted tissue expression, SIRPα antagonists may be better tolerated than agents targeting CD47, which is ubiquitously expressed in many tissues. Here, we investigated the therapeutic impact of monoclonal antibodies (mAbs) against CD47 and/or SIRPα on gastroenterological tumors in syngeneic immunocompetent mouse models.Methods: We used in vitro and in vivo phagocytosis assays in C57BL/6J (B6) mice to investigate anti‐CD47/SIRPα mAb effects on Hepa1‐6 and CMT93 originating from B6 mice. The influence of these mAbs on macrophage transmigration was also assessed. To investigate anti‐SIRPα mAb therapy‐induced inhibitory effects on sporadic colon cancer growth, we used a CDX2P9.5‐NLS Cre;APC⁺/FLOX (CPC‐APC) mouse model.Results: Systemic anti‐SIRPα mAb administration significantly increased Hepa1‐6 and CMT93 cell susceptibility to macrophage phagocytosis, both in vitro and in vivo. Conversely, similarly administered anti‐CD47 mAb did not promote macrophage phagocytosis of target cells, whereas cells incubated with anti‐CD47 mAb prior to inoculation were more susceptible to macrophage phagocytosis. In vitro cell migration assays revealed that binding with anti‐CD47 mAb inhibited macrophage transmigration. Anti‐SIRPα mAb treatment inhibited tumor progression in CPC‐APC mice and significantly improved overall survival. Anti‐CD47 mAb administration in vivo eliminated the phagocytosis‐promoting CD47 blockade effect, probably by inhibiting macrophage transmigration/chemotaxis. In contrast, anti‐SIRPα mAb exhibited enhanced macrophage phagocytic activity and marked anti‐tumor effects against gastroenterological malignancies.Conclusion: SIRPα mAb augmentation of macrophage phagocytic activity may represent an effective treatment strategy for human gastrointestinal tumors.

Gallbladder torsion is mainly associated with a floating gallbladder. From an anatomical perspective, laparoscopic cholecystectomy is a more optimal treatment than open cholecystectomy. An 84-year-old woman visited the Onomichi General Hospital because of progressive pain in the right upper quadrant of her abdomen. Physical examination revealed a positive Murphy sign and peritoneal irritation. Laboratory data demonstrated that inflammatory marker levels were increased. Abdominal ultrasonography showed that blood flow in the cystic artery was reduced and the gallbladder was swollen. Abdominal contrast-enhanced computerized tomography indicated that the swollen gallbladder was modestly enhanced and the fundus was displaced under the midline and detached from the gallbladder bed. The cystic duct was twisted. Magnetic resonance cholangiopancreatography showed that the root of the cystic duct was unclear and the extrahepatic bile duct had V-shaped distortion. The gallbladder neck showed a tapering interruption with the common biliary duct. We made a preoperative diagnosis of gallbladder torsion. Accordingly, emergency laparoscopic cholecystectomy was performed. The intraoperative findings included a dark swollen gallbladder that was twisted in the counterclockwise direction. The patient was discharged without any postoperative complications on day 7. Combined acute onset of abdominal pain with characteristic radiological findings made it possible to precisely diagnose gallbladder torsion. Laparoscopic cholecystectomy can be the gold standard treatment for gallbladder torsion after a preoperative diagnosis is made.