Biology subjects

Hayakawa, M.

Publications and source records attributed to Hayakawa, M..

10 recordsLinked to original sources

bioRxiv2026

A cuffed CRISPR guide RNA for microRNA activity-dependent genome editing

Cells in multicellular eukaryotic systems are diverse biological units, with characteristics and functions determined by their molecular profiles. CRISPR-Cas9 genome editing has been widely used across biology to modulate gene expression and study gene function. However, there is currently no versatile and scalable method for editing a cells genome in response to endogenous cellular signals. Here, we report the engineering of a CRISPR guide RNA that efficiently confers genome editing in response to the catalytic activity of a target microRNA (miRNA) within a cell. miRNAs are short non-coding RNAs that are widely conserved across eukaryotes and can cleave their target RNA through almost perfect base pairing. In mammals, miRNAs are largely involved in development and homeostasis as well as disease progression and developmental disorders. To leverage these properties for genome editing, we developed a cuffed guide RNA (cgRNA) which is composed of a permutated order of sequence domains from the commonly used single guide RNA (sgRNA). These permutated domains were then concatenated with a miRNA target sequence, yielding a warped guide RNA that is inactive until cleaved by a complementary miRNA. We demonstrated that cgRNA enabled efficient miRNA activity-dependent genome editing in human and mouse cell lines. Biochemical and structural analyses revealed three stages of inhibition of the CRISPR genome-editing pathway for unprocessed cgRNA. Utilizing a lentiviral library of cgRNAs containing miRNA targets covering mouse genome-wide miRNAs, we identified miRNA cleavage activities and their sequence specificities in mouse embryonic stem cells and during smooth muscle cell differentiation. Furthermore, we showed that endogenous mRNA expression could be irreversibly recorded into a DNA sequence using a cgRNA targeted by a synthetic miRNA repeat. cgRNA is a simple, robust, miRNA activity-gated genome editing system that could facilitate the development of cell state-specific genome editing, the mapping of miRNA activity and gene expression landscapes, and the recording of molecularly determined cell states during the long-term progression of multicellular systems.

bioengineering↗

bioRxiv2025

Therapeutic Knock-in Genome Editing Using Single AAV Vectors in Mouse Models of Inherited Liver Disease

Gene knock-in therapy has the potential to cure inherited liver diseases but is limited by low efficiency and delivery complexity. Here, we developed a single adeno-associated virus (AAV) vector system comprising a compact CRISPR effector, enAsCas12f, a guide RNA, and a donor template to enable therapeutic genome editing via non-homologous end joining (NHEJ). We targeted the system to the murine Alb locus and applied it to mouse models of hemophilia B, protein C (PC) deficiency, and ornithine transcarbamylase (OTC) deficiency. NHEJ-mediated knock-in showed higher efficiency than homology-directed repair, with successful therapeutic gene insertion in both neonatal and adult mice. The strategy restored plasma factor IX activity in hemophilia B (F9-/-) mice, prolonged survival of PC-deficient (Proc-/-) mice, and prevented hyperammonemia and weight loss in OTC-deficient (Otcspf-ash) mice upon high protein challenge. Importantly, gene integration was restricted to the liver, with no evidence of germline transmission. This compact, all-in-one AAV knock-in platform simplifies vector production, enables efficient delivery, and achieves reliable transgene expression in vivo. Our findings highlight the potential of liver-targeted knock-in genome editing as a transplant-independent treatment for neonatal-onset metabolic diseases, offering a clinically feasible path towards curative gene therapies for a wide range of monogenic liver disorders.

bioengineering↗

bioRxiv2024

Engineered coagulation factor VIII with enhanced secretion and coagulation potential for hemophilia A gene therapy

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). Here, we developed engineered human B-domain-deleted FVIIIs (FVIIISQs) with enhanced secretion and coagulation potential. Intracellular accumulation was markedly reduced in some engineered FVIIISQs, resulting in reduced unfolded protein responses. The administration of AAV vectors carrying engineered FVIIISQ to hemophilia A mice resulted in approximately eight-fold higher FVIII activity and four-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 supra-physiological increase in plasma FVIII activity at a dose one-thirtieth that of valoctocogene roxaparvovec (2 x 1012 vg/kg). The engineered FVIIISQ may thus provide stable, long-term therapeutic efficacy in AAV-mediated hemophilia A gene therapy even at low doses.

bioengineering↗

bioRxiv2024

Universal base editing for hemophilia B

The repair of pathological gene variants is an ultimate aim for treating genetic diseases; however, it is not practical to develop different therapeutic reagents for each of the many variants that can occur in a gene. Here, we investigated whether base editing to induce a gain-of-function variant in blood coagulation factor IX (FIX) can increase FIX activity as a treatment strategy 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 can become a universal genome editing strategy for hemophilia B.

bioengineering↗

bioRxiv2023

Cure of congenital purpura fulminans via expression of engineered protein C through neonatal genome editing in mice

Protein C (PC) 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. First, we generated an engineered activated PC to insert a self-cleaving peptide sequence between light and heavy chains. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of protein S in vitro. The adeno-associated virus (AAV) vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation factor V in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6 mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using AAV 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. These results suggest that the expression of the engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency. One Sentence SummaryEctopic expression of an engineered protein C via genome editing cures protein C deficiency in mice.

bioengineering↗

bioRxiv2022

Efficient Gene Transduction in Pigs and Macaques with the Engineered AAV Vector AAV.GT5 for Hemophilia B Gene Therapy

Gene therapy for hemophilia using adeno-associated virus (AAV) vectors allows long-term coagulation factor expression. 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, while in vivo transduction efficacy into the liver and the increase in coagulation factor IX (FIX) antigen following intravenous injection of these vectors were similar in PXB mice (chimeric mice with a humanized liver) and macaques. The 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 original AAV3B. The intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced vector transduction into the liver and reduced vector distribution to the kidney in pigs. In macaques, the intra-hepatic artery injection of AAV.GT5 yielded a comparable increase in FIX antigen with a one-third dosage of peripheral venous administration. Two of four macaques who received AAV.GT5 intravenously did not develop neutralizing antibodies (NAbs) against AAV.GT5, while AAV-Spark100 induced serotype-specific NAbs in all four macaques. The NAb produced after the administration was relatively specific to the serotype and less responsive to the other serotype. As a result, the administration of AAV.GT5 successfully boosted FIX expression in one animal previously given AAV-Spark100. Thus, AAV.GT5 has different biodistribution and immunogenic characteristics compared with AAV-Spark100, and the intra-hepatic vascular administration may lessen the vector dose and avoid vector distribution to other organs. Key PointsO_LIThe AAV.GT5 vector has a strong transduction efficacy in human hepatocytes but has a faster clearance after systemic administration. C_LIO_LIIntra-hepatic vascular administration of the AAV.GT5 vector is an effective liver transduction method for hemophilia gene therapy. C_LI

genetics↗

bioRxiv2022

Successful Liver transduction by Re-administration of Different Adeno-Associated Virus Vector Serotypes in Mice

Intravenous administration of adeno-associated virus (AAV) vector is a promising gene therapy approach for monogenic diseases. However, re-administration of the same AAV serotype is impossible due to the induction of anti-AAV neutralizing antibodies (NAbs). Here we examined the feasibility of re-administration of AAV vectors to change the serotypes. We administered AAV3B, AAV5, or AAV8 vectors targeting the liver of C57BL/6 mice intravenously, and then assessed the emergence of NAbs and the transduction efficacy with a second administration. For all serotypes, we confirmed that re-administration with the same serotype was not possible. Although the highest neutralizing activity of NAb was induced by AAV5; however, the NAbs elicited by AAV5 did not react with any other serotypes, resulting in success in re-administration with the other serotypes. The re-administration of AAV5 was also successful in all mice treated with AAV3B and AAV8. The effective secondary administration of AAV3B and AAV8 was observed in most mice treated with AAV8 and AAV3B, respectively. However, few mice developed NAbs cross-reactive with the other serotypes, especially the serotypes with close sequence homology. In summary, AAV vector administration induced NAbs relatively specific to the serotype administrated. Secondary administration of AAVs targeting liver transduction could be successfully achieved by switching AAV serotypes in mice.

molecular biology↗

bioRxiv2022

Beyond pathologist-level annotation of large-scale cancer histology for semantic segmentation using immunofluorescence restaining

Numerous cancer histopathology specimens have been collected and digitised as whole slide images over the past few decades. A comprehensive evaluation of the distribution of various cells in a section of tumour tissue can provide valuable information for understanding cancer and making accurate cancer diagnoses. Deep learning is one of the most suitable techniques to achieve these goals; however, the collection of large, unbiased training data has been a barrier to producing accurate segmentation models. Here, we developed a pipeline to generate SegPath, the largest annotation dataset that is over one order of magnitude larger than publicly available annotations, for the segmentation of haematoxylin and eosin (H&E)-stained sections for eight major cell types. The pipeline used H&E-stained sections that were destained and subsequently immunofluorescence-stained with carefully selected antibodies. The results showed that SegPath is comparable to, or significantly outperforms, conventional pathologist annotations. Moreover, we revealed that annotations by pathologists are biased toward typical morphologies; however, the model trained on SegPath can overcome this limitation. Our results provide foundational datasets for the histopathology machine learning community.

pathology↗

bioRxiv2022

Uncertainty quantification in the cerebral circulation simulation focusing on the collateral flow: Surrogate model approach with machine learning

Collateral circulation in the circle of Willis (CoW), closely associated with disease mechanisms and treatment outcomes, can be effectively investigated using one-dimensional-zero-dimensional hemodynamic simulations. As the entire cardiovascular system is considered in the simulation, it captures the systemic effects of local arterial changes, thus reproducing collateral circulation that reflects biological phenomena. The simulation facilitates rapid assessment of clinically relevant hemodynamic quantities under patient-specific conditions by incorporating clinical data. During patient-specific simulations, the impact of clinical data uncertainty on the simulated quantities should be quantified to obtain reliable results. However, as uncertainty quantification (UQ) is time-consuming and computationally expensive, its implementation in time-sensitive clinical applications is considered impractical. Therefore, we constructed a surrogate model based on machine learning using simulation data. The model accurately predicts the flow rate and pressure in the CoW in a few milliseconds. This reduced computation time enables the UQ execution with 100 000 predictions in a few minutes on a single CPU core and in less than a minute on a GPU. We performed UQ to predict the risk of cerebral hyperperfusion (CH), a life-threatening condition that can occur after carotid artery stenosis surgery if collateral circulation fails to function appropriately. We predicted the statistics of the postoperative flow rate increase in the CoW, which is a measure of CH, considering the uncertainties of arterial diameters, stenosis parameters, and flow rates measured using the patients clinical data. A sensitivity analysis was performed to clarify the impact of each uncertain parameter on the flow rate increase. Results indicated that CH occurred when two conditions were satisfied simultaneously: severe stenosis and when arteries of small diameter serve as the collateral pathway to the cerebral artery on the stenosis side. These findings elucidate the biological aspects of cerebral circulation in terms of the relationship between collateral flow and CH. Author summaryCerebral arteries generate a ring-like network that provides alternative routes for blood supply in the case of carotid artery stenosis. This collateral circulation is closely associated with the potential risk of stroke and treatment outcomes in patients with stenosis. In this study, we propose a method to elucidate the cerebral circulation of individual patients using a blood flow simulation that incorporates the patients clinical data. A key feature of our approach is its capability to obtain the probability of the different outputs using simulation, considering the uncertainty of patient conditions. Although this capability is essential for obtaining reliable results, the process is time-consuming and requires numerous computer resources. We solved this problem by combining the blood flow simulation with machine learning to perform predictions 43 000 times faster than conventional simulations. We applied the proposed method to predict cerebral circulation following surgery in three patients with stenosis and verified that the method can assess the surgical risk almost in real-time, even on a desktop computer. Additionally, extensive prediction results (100 000 cases for each patient) obtained using this method clarify the relationship between collateral circulation and life-threatening surgical outcomes.

bioengineering↗

bioRxiv2019

Polar pattern formation induced by contact following locomotion in a multicellular system

Biophysical mechanisms underlying collective cell migration of eukaryotic cells have been studied extensively in recent years. One paradigm that induces cells to correlate their motions is contact inhibition of locomotion, by which cells migrating away from the contact site. Here, we report that tail-following behavior at the contact site, termed contact following locomotion (CFL), can induce a non-trivial collective behavior in migrating cells. We show the emergence of a traveling band showing polar order in a mutant Dictyostelium cell that lacks chemotactic activity. The traveling band is dynamic in the sense that it continuously assembled at the front of the band and disassembled at the back. A mutant cell lacking cell adhesion molecule TgrB1 did not show both the traveling band formation and CFL. We thus conclude that CFL is the cell-cell interaction underlying the traveling band formation. We then develop an agent-based simulation with CFL, which shows the role of CFL in the formation of traveling band. We further show that the polar order phase consists of subpopulations that exhibit characteristic transversal motions with respect to the direction of band propagation. These findings describe a novel mechanism of collective cell migration involving cell-cell interactions capable of inducing traveling band with polar order.

biophysics↗