柏倉 裕志

Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Although PAM-relaxed Cas9 variants have been developed, expanded targeting is often accompanied by compromised target specificity. Here, we report the rational engineering of eSaCas9-NNG, a SaCas9 variant that recognizes relaxed NNG PAMs while maintaining high target fidelity, thereby overcoming a fundamental trade-off in Cas9-based genome editing. eSaCas9-NNG efficiently induces indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice, with editing efficiencies comparable to those of other PAM-relaxed nucleases, including SpRY, SpG, and iGeoCas9, but with reduced off-target activity. We further determine the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for its relaxed PAM recognition, improved target specificity, and nuclease activation. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.

The major challenges of gene therapy for hemophilia A using adeno-associated virus (AAV) vectors are reducing vector doses and the long-term maintenance of stable factor VIII (FVIII). In this study, we developed engineered human B-domain-deleted FVIIIs (FVIIISQ) with enhanced secretion and coagulation potential. Intracellular accumulation was markedly reduced in some engineered FVIIISQ, resulting in reduced unfolded protein responses. The administration of AAV vectors carrying engineered FVIIISQ to hemophilia A mice resulted in ∼8-fold higher FVIII activity and 4-fold higher FVIII antigen levels compared with wild-type FVIIISQ administration. The specific FVIII activity of the engineered FVIIISQ was 3.6 times higher than that of the wild-type FVIIISQ, and its binding to activated coagulation factor IX was significantly enhanced, which is supported by the structural analysis. In macaques, the administration of AAV5 vector carrying the engineered FVIIISQ without CpG sequences resulted in a supraphysiological increase in plasma FVIII activity at a dose one-thirtieth that of valoctocogene roxaparvovec (2 × 1012 vector genome per kg). The engineered FVIIISQ may thus provide stable, long-term therapeutic efficacy in AAV-mediated hemophilia A gene therapy even at low doses.

The repair of pathological gene variants is an ultimate aim for treating genetic diseases; however, the development of different therapeutic reagents for each of the many variants that can occur in a gene may not be scalable. Here, we investigated whether base editing to introduce a gain-of-function variant in blood coagulation factor IX (FIX) can increase FIX activity as a targeted therapeutic approach for hemophilia B. We engineered a G:C to A:T substitution at c.1151 of F9 by cytosine base editing to generate R338Q, known as the Shanghai F9 variant, which markedly potentiates coagulation factor activity. An adeno-associated virus vector harboring the base editor converted more than 60% of the target G:C to A:T and increased FIX activity in HEK293 cells harboring patient-derived F9 variants, as well as in knock-in mice harboring a human F9 cDNA. Furthermore, administration of lipid nanoparticles embedded with the base editor mRNA and gRNA increased FIX activity in mice. These data indicate that cytosine base editing to generate R338Q in FIX is a broadly applicable genome editing approach for hemophilia B with residual FIX activity.

BACKGROUND: PC (protein C) 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. METHODS: We generated an engineered APC (activated PC) to insert a furin-cleaving peptide sequence between light and heavy chains. The engineered PC was expressed in the liver of mice using an adeno-associated virus vector or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated genome editing using an adeno-associated virus vector in vivo. RESULTS: The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of PS (protein S) in vitro. The adeno-associated virus vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation FV (factor V) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6J mice. The insertion of EGFP (enhanced green fluorescent protein) sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using adeno-associated virus 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. CONCLUSIONS: These results suggest that the expression of engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.

Abstract A2 domain dissociation in activated factor VIII (FVIIIa) results in reduced activity. Previous studies demonstrated that some FVIII mutants (D519V/E665V and K1813A) with delayed A2 dissociation enhanced coagulation potential. We speculated, therefore, that FVIII encompassing a combination of these mutations might further enhance coagulant activity. The aim was to assess the D519V/E665V/K1813A-FVIII mutation as a gain of function. The FVIII mutants, D519V/E665V/K1813A, D519V/E665V, and K1813A were expressed in a baby hamster kidney cell system, and global coagulation potential of these mutants was compared with wild-type (WT) FVIII in vitro and in hemophilia A mice in vivo. Kinetic analyses indicated that the apparent Kd for FIXa on the tenase assembly with D519V/E665V and D519V/E665V/K1813A mutants were lower, and that the generated FXa for D519V/E665V/K1813A was significantly greater than WT-FVIII. WT-FVIII activity after thrombin activation increased by ∼12-fold within 5 minutes, and returned to initial levels within 30 minutes. In contrast, The FVIII-related activity of D519V/E665V/K1813A increased further with time after thrombin activation, and showed an ∼25-fold increase at 2 hours. The A2 dissociation rate of D519V/E665V/K1813A was ∼50-fold slower than the WT in a 1-stage clotting assay. Thrombin generation assays demonstrated that D519V/E665V/K1813A (0.125 nM) exhibited coagulation potential comparable with that of the WT (1 nM). In animal studies, rotational thromboelastometry and tail-clip assays showed that the coagulation potential of D519V/E665V/K1813A (0.25 μg/kg) was equal to that of the WT (2 μg/kg). FVIII-D519V/E665V/K1813A mutant could provide an approximately eightfold increase in hemostatic function of WT-FVIII because of increased FVIIIa stability and the association between FVIIIa and FIXa.

The importance of genetic diagnosis for patients with hemophilia has been recently demonstrated. However, the pathological variant cannot be identified in some patients. Here, we aimed to identify the pathogenic intronic variant causing hemophilia A using induced pluripotent stem cells (iPSCs) from patients and genome editing. We analyzed siblings with moderate hemophilia A and without abnormalities in the F8 exon. Next-generation sequencing of the entire F8 revealed 23 common intron variants. Variant effect predictor software indicated that the deep intronic variant at c.5220-8563A>G (intron 14) might act as a splicing acceptor. We developed iPSCs from patients and used genome editing to insert the elongation factor 1α promoter to express F8 messenger RNA (mRNA). Then, we confirmed the existence of abnormal F8 mRNA derived from aberrant splicing, resulting in a premature terminal codon as well as a significant reduction in F8 mRNA in iPSCs due to nonsense-mediated RNA decay. Gene repair by genome editing recovered whole F8 mRNA expression. Introduction of the intron variant into human B-domain-deleted F8 complementary DNA suppressed factor VIII (FVIII) activity and produced abnormal FVIII lacking the light chain in HEK293 cells. Furthermore, genome editing of the intron variant restored FVIII production. In summary, we have directly proven that the deep intronic variant in F8 results in aberrant splicing, leading to abnormal mRNA and nonsense-mediated RNA decay. Additionally, genome editing targeting the variant restored F8 mRNA and FVIII production. Our approach could be useful not only for identifying causal variants but also for verifying the therapeutic effect of personalized genome editing.

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.

<title>Abstract</title>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 <italic>F8</italic> 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 <italic>F8</italic> 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.