PB0985 - Cure of Congenital Purpura Fulminant by Neonatal Genome Editing to Express an Engineered Protein C in Mice

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

TT

Tomoki Togashi

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

Co-Author(s)

NB

Nemekhbayar Baatartsogt

Postdoctoral Fellow
Department of Biochemistry, Jichi Medical University School of Medicine
Shimotsuke, Tochigi, Japan
YN

Yasumitsu Nagao

Associate Professor
Center for Experimental Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan
YK

Yuji Kashiwakura, PhD (he/him/his)

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

Morisada Hayakawa

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

Nobuhiko Kamoshita

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

Takafumi Hiramoto

Research Associate
Department of Biochemistry, Jichi Medical University School of Medicine
Shimotsuke, Tochigi, Japan
TF

Takayuki Fujiwara

Assistant Professor
Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan
Eriko Morishita, MD, PhD

Professor
Department of Clinical Laboratory Science, Division of Health Sciences, Graduate School of Medical Science, Kanazawa University
Kanazawa, Ishikawa, Japan
ON

Osamu Nureki

Professor
Department of Biological Sciences, Graduate School of Science, The University of Tokyo
Bunkyo Ward, Tokyo, Japan
Tsukasa Ohmori, MD, PhD (he/him/his)

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

Background: Protein C (PC) is a vitamin K-dependent plasma anticoagulant encoded by PROC produced from the liver. The abnormality in both PROC alleles results in neonatal purpura fulminans—a fatal systemic thrombotic disorder.

Aims: We aimed to develop a genome editing treatment to cure congenital PC deficiency.

Methods: We designed several engineered PCs to release as activated form, and obtained the engineered PC proteins from HEK293 cells stably expressing the cDNAs. The PC activity was measured by Berichrom PROTEIN C by an automated coagulation analyzer. Adeno-associated virus (AAV) vector was applied to assess the anticoagulant activity of the engineered PC and genome editing in mice. Thrombus formation induced by reactive oxygen species in mice was observed with intravital microscopy. PC-deficient mice were generated by the genome editing of mouse zygotes.

Results: We identified the engineered PC which could be released in its activated form because PC activity was detected without activation by snake venom. The engineered PC significantly prolonged the plasma coagulation time independent of the cofactor activity of protein S in vitro. The 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 (FV) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6 mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo. PC-deficient mice treated with genome editing to express the engineered PC exhibited normal thrombus formation induced by laser injury.

Conclusion(s): The engineered PC can be released as an activated form and protect pathological thrombus formation in vivo, depending on the inhibition of activated FV. The expression of the engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.