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Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galβ1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%–1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. 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, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This 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 capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination.

Abstract 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.

Adeno-associated virus (AAV) vectors are promising modalities of gene therapy to address unmet medical needs. However, anti-AAV neutralizing antibodies (NAbs) hamper the vector-mediated therapeutic effect. Therefore, NAb prevalence in the target population is vital in designing clinical trials with AAV vectors. Hence, updating the seroprevalence of anti-AAV NAbs, herein we analyzed sera from 100 healthy individuals and 216 hemophiliacs in Japan. In both groups, the overall seroprevalence against various AAV serotypes was 20%–30%, and the ratio of the NAb-positive population increased with age. The seroprevalence did not differ between healthy participants and hemophiliacs and was not biased by the concomitant blood-borne viral infections. The high neutralizing activity, which strongly inhibits the transduction with all serotypes in vitro, was mostly found in people in their 60s or of older age. The multivariate analysis suggested that “60s or older age” was the only independent factor related to the high titer of NAbs. Conversely, a large proportion of younger hemophiliacs was seronegative, rendering them eligible for AAV-mediated gene therapy in Japan. Compared with our previous study, the peak of seroprevalences has shifted to older populations, indicating that natural AAV exposure in the elderly occurred in their youth but not during the last decade.

Abstract IκBζ is a transcriptional regulator that augments inflammatory responses from the Toll-like receptor or interleukin signaling. These innate immune responses contribute to the progression of nonalcoholic fatty liver disease (NAFLD); however, the role of IκBζ in the pathogenesis of NAFLD remains elusive. We investigated whether IκBζ was involved in the progression of NAFLD in mice. We generated hepatocyte-specific IκBζ-deficient mice (Alb-Cre; Nfkbiz fl/fl ) by crossing Nfkbiz fl/fl mice with Alb-Cre transgenic mice. NAFLD was induced by feeding the mice a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). CDAHFD-induced IκBζ expression in the liver was observed in Nfkbiz fl/fl mice, but not in Alb-Cre; Nfkbiz fl/fl mice. Contrary to our initial expectation, IκBζ deletion in hepatocytes accelerated the progression of NAFLD after CDAHFD treatment. Although the increased expression of inflammatory cytokines and apoptosis-related proteins by CDAHFD remained unchanged between Nfkbiz fl/fl and Alb-Cre; Nfkbiz fl/fl mice, early-stage steatosis of the liver was significantly augmented in Alb-Cre; Nfkbiz fl/fl mice. Overexpression of IκBζ in hepatocytes via the adeno-associated virus vector attenuated liver steatosis caused by the CDAHFD in wild-type C57BL/6 mice. This preventive effect of IκBζ overexpression on steatosis was not observed without transcriptional activity. Microarray analysis revealed a correlation between IκBζ expression and the changes of factors related to triglyceride biosynthesis and lipoprotein uptake. Our data suggest that hepatic IκBζ attenuates the progression of NAFLD possibly through the regulation of the factors related to triglyceride metabolism.

Most gene therapy clinical trials that systemically administered adeno-associated virus (AAV) vector enrolled only patients without anti-AAV-neutralizing antibodies. However, laboratory tests to measure neutralizing antibodies varied among clinical trials and have not been standardized. In this study, we attempted to improve the sensitivity and reproducibility of a cell-based assay to detect neutralizing antibodies and to determine the detection threshold to predict treatment efficacy. Application of the secreted type of NanoLuc and AAV receptor-expressing cells reduced the multiplicity of infection (MOI) for AAV transduction and improved the sensitivity to detect neutralizing antibodies with a low coefficient of variation, whereas the detection threshold could not be improved by the reduction of MOI to

Abstract Coagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin− and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin− in liver sinusoidal endothelial cells.

We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.

Background: Anticoagulant activity and blood pressure increase in the morning. The aim of this study was to evaluate changes of anticoagulant activity, blood pressure and target organ damage in patients with nonvalvular atrial fibrillation (AF) given combination treatment with Xa inhibitor and antihypertensive agent. Methods: We enrolled 72 patients with nonvalvular AF. Rivaroxaban (10–15 mg) was continuously administered once daily over 8 weeks (study period I). For subjects (n = 50) who exhibited uncontrolled morning hypertension (home systolic blood pressure [SBP]≥125 mmHg) at the end of study period I (at 8 weeks), nifedipine CR (20–40 mg) was added at bedtime, and rivaroxaban administration was continued an additional 8 weeks. We assessed prothrombin fragment 1 + 2 (optimal range: 69–229 pmol/L) and D-dimer (negative D-dimer measurement:

Abstract Psychoneuroimmunological studies have clearly demonstrated that both cellular and humoral immunity are related to major depression. Soluble ST2 is regarded as a key molecule regulating immune system as well as cell proliferation. Indeed, soluble ST2 is reported to reduce IL-33-induced IL-6 and TNF-α production in macrophages and IL-33-induced IL-5 and IL-13 production in type 2 innate lymphoid cells. Elevated serum concentrations of soluble ST2 have been reported in patients with neuropsychiatric disorders, suggesting pathophysiological roles of soluble ST2 in behavioral phenotypes. Nevertheless, the relation between soluble ST2 and depressive behavior remain to be uncovered. To complement this point, we performed broad behavioral phenotyping, utilizing transgenic mice with a high concentration of serum ST2 in the present study. Soluble ST2 overexpression mice (ST2 Tg mice) were generated on a C3H/HeJ background. ST2 Tg mice crossed onto the BALB/c genetic background were used. Before starting tests, each mouse was observed in a clean cage for a general health check and neurological screening tests. In Experiment I, comprehensive behavioral phenotyping was performed to reveal the role of soluble ST2 on sensorimotor functions, anxiety-like behaviors, depression-like behaviors, social behaviors, and learning and memory functions. In Experiment II, to confirm the role of soluble ST2 on depression-like behaviors, a depression test battery (two bottle choice test, forced swimming test, and tail suspension test) was applied. The general health check indicated good general health and normal gross appearance for ST2 Tg mice. Further, the neurological reflexes of all the mice were normal. We found that soluble ST2 overexpression resulted in decreased social interaction. Moreover, depression-like behaviors of ST2 Tg mice were observed in two well-established behavioral paradigms, the forced swimming test and the tail suspension test. Nevertheless, hedonic reaction to sucrose was observed in ST2 Tg mice similar to WT mice. These results suggest the depression in the ST2 Tg mice. In conclusion, through a series of experiments, we established the animal model for assessing role of soluble ST2 in neuropsychiatric disorders, and revealed the possible involvement of soluble ST2 in depressive behavior.

Abstract Background Acquired von Willebrand syndrome (aVWS) is a rare bleeding disorder with laboratory findings similar to those of congenital von Willebrand disease (VWD). Patients with aVWS may require prophylactic treatment to prevent excessive bleeding following surgery. To our knowledge, to date, there have been no reports on perioperative management for breast cancer patients with aVWS. Case presentation A 60-year-old woman with breast cancer was diagnosed with aVWS due to polycythemia vera. Pre-operative laboratory testing showed a high platelet count and low von Willebrand factor (VWF) activity. The VWF activity did not improve despite an attempt to suppress platelet count with hydroxyurea. Therefore, we decided to perioperatively supplement with plasma-derived factor VIII (FVIII) containing von Willebrand factor (FVIII/VWF concentrates) to perform curative surgery for breast cancer safely. Consequently, the patient did not develop hemorrhage during/after surgery and was discharged on postoperative day 7, as planned, without problems. Conclusions For a patient with aVWS, which carries a high risk of hemorrhage during the perioperative period, initiation of appropriate management like supplementation of FVIII/VWF concentrates might enable safe curative surgery for breast cancer, and collaboration with the hematology department is critical.

Abstract Background Although prasugrel exerts stronger antiplatelet effects compared with clopidogrel, the factors affecting platelet reactivity under prasugrel have not been fully determined. This study aimed to find the novel mechanistic differences between two thienopyridines and identify the factor that influence platelet reactivity to each drug. Methods Forty patients with stable angina who underwent elective percutaneous coronary intervention were randomly assigned to receive either prasugrel (20 mg) or clopidogrel (300 mg) as a loading dose. Platelet function (light transmission, laser light scattering, and vasodilator-stimulated phosphoprotein phosphorylation) and plasma active metabolite levels were measured after the loading dose. Results Prasugrel consistently inhibited adenosine diphosphate receptor P2Y12 signalling to abolish amplification of platelet aggregation. Prasugrel abolished even small platelet aggregates composed of less than 100 platelets. On the other hand, clopidogrel inhibited large aggregates but increased small and medium platelet aggregates. Diabetes was the only independent variable for determining antiplatelet effects and active metabolite concentration of prasugrel, but not clopidogrel. Sleep-disordered breathing was significantly correlated with platelet reactivity in patients who had clopidogrel. Conclusions Prasugrel efficiently abolishes residual P2Y12 signalling that causes small platelet aggregates, but these small aggregates are not inhibited by clopidogrel. Considering the differential effect of diabetes on antiplatelet effects between these two drugs, the pharmacokinetics of prasugrel, other than cytochrome P450 metabolism, might be affected by diabetes. Trial registration UMIN-CTR UMIN000017624 , retrospectively registered 21 May 2015.

Abstract Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.

Although bleeding is a common complication of surgery, routine laboratory tests have been demonstrated to have a low ability to predict perioperative bleeding. Better understanding of hemostatic function during surgery would lead to identification of high-risk patients for bleeding. Here, we aimed to elucidate hemostatic mechanisms to determine perioperative bleeding. We prospectively enrolled 104 patients undergoing cervical spinal surgery without bleeding diathesis. Blood sampling was performed just before the operation. Volumes of perioperative blood loss were compared with the results of detailed laboratory tests assessing primary hemostasis, secondary hemostasis, and fibrinolysis. Platelet aggregations induced by several agonists correlated with each other, and only two latent factors determined inter-individual difference. Platelet aggregability independently determined perioperative bleeding. We also identified low levels of plasminogen-activator inhibitor-1 (PAI-1) and α2-plasmin inhibitor to be independent risk factors for intraoperative and postoperative bleeding, respectively. Most important independent factor to determine postoperative bleeding was body weight. Of note, obese patients with low levels of PAI-1 became high-risk patients for bleeding during surgery. Our data suggest that bleeding after surgical procedure may be influenced by inter-individual differences of hemostatic function including platelet function and fibrinolysis, even in the patients without bleeding diathesis.

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.

Body weight is tightly regulated by food intake and energy dissipation, and obesity is related to decreased energy expenditure (EE). Herein, we show that nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2, autotaxin) is an adipose-derived, secreted enzyme that controls adipose expansion, brown adipose tissue (BAT) function, and EE. In mice, Enpp2 was highly expressed in visceral white adipose tissue and BAT and is downregulated in hypertrophied adipocytes/adipose tissue. Enpp2+/- mice and adipocyte-specific Enpp2 knockout mice fed a high-fat diet showed smaller body weight gains and less insulin resistance than control mice fed the same diet. BAT was functionally more active and EE was increased in Enpp2-deficient mice. In humans, ENPP2 expression in subcutaneous fat and ENPP2 levels in serum were reduced in obese subjects. Taken together, our results establish ENPP2 as an adipose-derived, secreted enzyme that regulates adipose obesity and systemic metabolism. They also suggest ENPP2 could be a useful therapeutic target for the treatment of metabolic disease. [ABSTRACT FROM AUTHOR]

IkBζ (encoded by the Nfkbiz) is a member of the nuclear IkB family, which is involved in the expression of secondary response genes based on signals from TLR or IL-1R. ST2L, an IL-33R, is a member of the IL-1R family and abundantly expressed in tissueresident immune cells, such as mast cells and innate lymphoid cells; however, its downstream signaling pathway remains unelucidated. In this study, we examined the role of IkBz in ST2L-mediated cytokine and chemokine production in mast cells. Murine bone marrow cells were differentiated ex vivo into bone marrow-derived mast cells (BMMCs). The treatment of BMMCs with IL-33 transiently induced robust IkBζ expression. Of the 40 cytokines and chemokines examined using a cytokine and chemokine array, the concentrations of IL-6, IL-13, CCL2, CCL3, and TNF-a in the supernatant were augmented by IL-33. The deletion of IkBz in BMMCs resulted in a significant reduction of the production of these mediators and the expression of their mRNA. NF-kB p50 but not p65 translocated to the nucleus by IL-33 and was not affected by the deletion of IkBζ. However, induction of IkBz and the resultant cytokine and chemokine productions were significantly inhibited by pretreatment with an NF-kB inhibitor. The deletion of IkBζ did not affect the phosphorylation of ERK, p38 MAPK, or JNK by IL-33, and the treatment with inhibitors of these mitogen-activated kinases failed to abolish the expression of Nfkbiz. Our findings suggest that IkBz augments IL-33-dependent cytokine and chemokine production in BMMCs through the action of NF-kB. [ABSTRACT FROM AUTHOR]

Intravital visualization of thrombopoiesis revealed that formation of proplatelets, which are cytoplasmic protrusions in bone marrow megakaryocytes (MKs), is dominant in the steady state. However, it was unclear whether this is the only path to platelet biogenesis. We have identified an alternative MK rupture, which entails rapid cytoplasmic fragmentation and release of much larger numbers of platelets, primarily into blood vessels, which is morphologically and temporally different than typical FasL-induced apoptosis. Serum levels of the inflammatory cytokine IL-1α were acutely elevated after platelet loss or administration of an inflammatory stimulus to mice, whereas the MK-regulator thrombopoietin (TPO) was not elevated. Moreover, IL-1α administration rapidly induced MK rupture-dependent thrombopoiesis and increased platelet counts. IL-1α-IL-1 R1 signaling activated caspase-3, which reduced plasma membrane stability and appeared to inhibit regulated tubulin expression and proplatelet formation, and ultimately led to MK rupture. Collectively, it appears the balance between TPO and IL-1α determines the MK cellular programming for thrombopoiesis in response to acute and chronic platelet needs. [ABSTRACT FROM AUTHOR]