PB0210 - Development of modified factor VIII for effective hemophilia A gene therapy

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

YK

Yuji Kashiwakura, PhD

Assistant Professor
Department of Biochemistry, School of Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan

Co-Author(s)

Yuto Nakajima, Md.PhD

Assistant professor
Department of Pediatrics
Kashihara, Nara, Japan
TL

Tiago Lopes

Scientist
Nezu Life Sciences
Setagaya, Tokyo, Japan
Nemekhbayar Baatartsogt, MD, PhD

Assistant Professor
Department of Biochemistry, School of Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan
NK

Nobuhiko Kamoshita

Assistant Professor
Department of Biochemistry, School of Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan
Morisada Hayakawa, PhD

Assistant Professor
Department of Biochemistry, School of Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan
YK

Yuko Katakai

veterinarian
The Corporation for Production and Research of Laboratory Primates
Tsukuba, Ibaraki, Japan
KN

Keiji Nogami

Professor
Nara Medical University, Nara, Japan
Nara, Nara, Japan
Tsukasa Ohmori

Professor
Department of Biochemistry, School of Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan

Background: Gene therapy for hemophilia A using adeno-associated virus (AAV) vectors requires a high dose vector and has not observed the stable factor VIII (FVIII) expression over the long term. One reason for the attenuation of FVIII expression is believed to be the unfolded protein response (UPR) due to ectopic expression of FVIII in hepatocytes.

Aims: This study aims to develop the engineered FVIII with high activity and lower UPR for effective AAV vector-mediated hemophilia A gene therapy.

Methods: We evaluated the expression, activity, and UPR of FVIII in Huh7 cells. FVIII expression in vivo was evaluated by administration of AAV vectors harboring FVIII to hemophilia A mice and macaques.

Results: We developed several modified human B-domain-deleted FVIII (FVIIISQ) (Ver.1-4) with high FVIII activity (FVIII:C) by multiple amino acid substitutions. Intracellular accumulation was remarkably reduced in FVIIISQ Ver.2 and Ver.4, resulted in the reduction of UPR in the human hepatocellular cell line Huh-7 with the transient expression. Administration of AAV vectors carrying FVIIISQ Ver.4 to hemophilia A mice resulted in about 8-fold higher FVIII:C in one stage clotting assay and 3-times FVIII antigen compared with those with wild type. The specific FVIII:C of FVIIISQ Ver.4 protein was 3.6-fold higher than that of the wild FVIIISQ and the binding to activated coagulation factor IX and coagulation factor X was significantly enhanced. The amino substitutions in FVIIISQ Ver.4 did not elicit antigenicity against major human HLAs in in-silico analysis. Finally, the administration of AAV8 vector carrying FVIIISQ Ver.4 without CpG sequences in macaques resulted in about 600% increase in FVIII:C at a low vector dose (2 x 10^12 vg/kg).

Conclusion(s): We identified the modified FVIIISQ with higher activity and lower UPR. The gene therapy with the modified FVIIISQ may provide stable and long-term therapeutic efficacy even at a low dose in AAV-mediated hemophilia A gene therapy.