Biology subjects

Baatartsogt, N.

Publications and source records attributed to Baatartsogt, N..

6 recordsLinked to original sources

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↗