Hemophilia is a congenital hemorrhagic disease caused by genetic abnormalities in coagulation factor VIII or factor IX. Current conventional therapy to prevent bleeding requires frequent intravenous ...injections of coagulation factor concentrates from early childhood. Accordingly, gene therapy for hemophilia remains an exciting future prospect for patients and their families, due to its potential to cure the disease through a one-time treatment. After a series of successes in basic research, recent clinical trials have demonstrated clear efficacy of gene therapy for hemophilia using adeno-associated virus (AAV) vectors. Although this is likely to alter the paradigm of hemophilia care in the near future, it will be important to overcome immune responses against AAV. Gene therapy for hemophilia cannot be given to patients with anti-AAV capsid-neutralizing antibodies, and cellular immunity with CD8
+
T cells should be controlled for sustained expression. Furthermore, long-term therapeutic effects should be closely observed because of the failure of the AAV vector genome to replicate during cell division. This review focuses on the basis of gene therapy, current successes of clinical trials, and the future direction of hemophilia gene therapy.
Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene ...therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.
Gene therapy is an opportunity for haemophilia patients to receive a one‐time treatment and have lasting factor levels for years or decades instead of dependence on repeated administration within ...short intervals and on sustained supply of drug. Great strides have been made in the development of gene therapy for haemophilia in the last decade. Adeno‐associated virus (AAV) vector–mediated gene transfer in haemophilia A and B has entered the phase III trial stage. Gene transfer by lentiviral vector or gene editing technologies using factor VIII (FVIII) or IX (FIX) genes are now entering clinical evaluation. It is expected that the first FVIII and FIX gene therapy products will soon be approved and distributed in major markets. Global access to gene therapy is a critical goal. This review presents new and ongoing efforts towards this goal in countries other than North America and Europe. In Japan, researchers, regulators and funders have established a promising gene therapy development platform for multiple diseases including haemophilia. Decades of scientific and clinical research in haemophilia gene therapy in China have led to a recently registered clinical trial of AAV‐mediated gene therapy for haemophilia B. Other countries are in earlier phases of building gene therapy programmes or participate in international trials. A phase 2 feasibility trial of AAV‐mediated FIX gene therapy in low‐ and middle‐income countries aims to demonstrate that gene therapy could become available in resource‐constrained socio‐economic settings. The different strategies for establishing gene therapy provide opportunities for closing the global gap in haemophilia care.
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.
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 CD31
high
, CD146
high
, 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 CD31
high
, CD146
high
, Lyve1
+
, CLEC-2
+
, and podoplanin
−
in liver sinusoidal endothelial cells.
Background
Intravenous administration of adeno‐associated virus (AAV) vectors is a promising gene therapy approach for monogenic diseases. However, re‐administration of the same AAV serotype is ...impossible because of the induction of anti‐AAV neutralizing antibodies (NAbs). Here, we examined the feasibility of re‐administrating AAV vector serotypes different from the initial AAV vector serotype.
Methods
Liver‐targeting AAV3B, AAV5, and AAV8 vectors were intravenously injected in C57BL/6 mice, and the emergence of NAbs and the transduction efficacy following re‐administration were evaluated.
Results
For all serotypes, re‐administration of the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5, anti‐AAV5 NAbs did not react with other serotypes, resulting in successful re‐administration with the other serotypes. AAV5 re‐administration was also successful in all mice treated with AAV3B and AAV8. Effective secondary administration of AAV3B and AAV8 was observed in most mice initially administrated AAV8 and AAV3B, respectively. However, few mice developed NAbs cross‐reacting with the other serotypes, especially those with close sequence homology.
Conclusions
In summary, AAV vector administration induced NAbs relatively specific to the administrated serotype. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice.
Systemic administration of AAVs targeting liver transduction induced high titer serotype‐specific neutralizing antibodies in mice. Here, the secondary administration was successfully achieved by switching AAV serotypes. Considering the re‐administration of AAV vectors to patients who have failed to respond to initial gene therapy or lost therapeutic effect over time, the development of several AAV preparations is crucial for targeting one disease.
Current criteria for early diagnosis of coagulopathy in sepsis are limited. We postulated that coagulopathy is already complicated with sepsis in the initial phase, and severe coagulopathy or ...disseminated intravascular coagulation (DIC) becomes overt after progressive consumption of platelet and coagulation factors. To determine early diagnostic markers for severe coagulopathy, we evaluated plasma biomarkers for association with subsequent development of overt DIC in patients with sepsis.
A single-center, prospective observational study was conducted in an adult ICU at a university hospital. Plasma samples were obtained from patients with sepsis at ICU admission. Fourteen biomarkers including global markers (platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen and fibrin degradation product (FDP)); markers of thrombin generation (thrombin-antithrombin complex (TAT) and soluble fibrin); markers of anticoagulants (protein C (PC) and antithrombin); markers of fibrinolysis (plasminogen, α2-plasmin inhibitor (PI), plasmin-α2-PI complex, and plasminogen activator inhibitor (PAI)-1); and a marker of endothelial activation (soluble E-selectin) were assayed. Patients who had overt DIC at baseline were excluded, and the remaining patients were followed for development of overt DIC in 5 days, and for mortality in 28 days.
A total of 77 patients were enrolled, and 37 developed overt DIC within the following 5 days. Most patients demonstrated hemostatic abnormalities at baseline with 98.7% TAT, 97.4% FDP and 88.3% PC. Most hemostatic biomarkers at baseline were significantly associated with subsequent development of overt DIC. Notably, TAT, PAI-1 and PC discriminated well between patients with and without developing overt DIC (area under the receiver operating characteristic curve (AUROC), 0.77 (95% confidence interval, 0.64 to 0.86); 0.87 (0.78 to 0.92); 0.85 (0.76 to 0.91), respectively), and using the three together, significantly improved the AUROC up to 0.95 (vs. TAT, PAI-1, and PC). Among the significant diagnostic markers for overt DIC, TAT and PAI-1 were also good predictors of 28-day mortality (AUROC, 0.77 and 0.81, respectively).
Severe coagulation and fibrinolytic abnormalities on ICU admission were associated with subsequent development of overt DIC. A single measurement of TAT, PAI-1, and PC activity could identify patients with ongoing severe coagulopathy, early in the course of sepsis.
SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) ...vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.
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•Cryo-EM structures of compact AsCas12f in complex with sgRNA and target DNA•AsCas12f engineering by structural analysis and deep mutational scanning methods•Activities of enAsCas12f variants, SpCas9, and AsCas12a variants are comparable•enAsCas12f effectors have been harnessed as a compact transcriptional activation tool
A high-throughput approach, combining deep mutational scanning and structure-informed design, generated miniature AsCas12f variants with enhanced activity, enabling efficient knock-in/knock-out activities and transcriptional activation in mice using an all-in-one AAV vector.
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.
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: <1.0 μg/mL).
Results: The percentage of patients with optimal-range prothrombin fragment 1 + 2 was significantly increased at 4 weeks compared to baseline (70.8% vs. 86.1%, p = .033). In period II, office and home morning SBP were reduced at 12 compared to 8 weeks (office SBP: 135.2 ± 15.7 vs. 125.6 ± 18.4mmHg, p < .001; home morning SBP: 133.5 ± 10.5 vs. 119.9 ± 12.1mmHg, p<.001).The percentage of patients with negative D-dimer was increased at 8 weeks compared to baseline (92% vs. 100%, p = .044), and remained at 100% at 16 weeks.
Conclusions: Xa inhibitor therapy improved anticoagulant activity, and additional antihypertensive therapy maintained the anticoagulant activity in patients with nonvalvular AF.
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