PB0165 - Gene correction of human hemophilia B mutation by PAM-flexible Cas9-mediated base-editing approach

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Co-Author(s)

TH

Takafumi Hiramoto

Research Associate
Department of Biochemistry, Jichi Medical University School of Medicine
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
NB

Nemekhbayar Baatartsogt

Postdoctoral Fellow
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
TA

Tomoyuki Abe

Assisitant Professor
Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan
Hiroshi Inaba, PhD

Associate Professor
Department of Laboratory Medicine, Tokyo Medical University
Shinjuku-ku, Tokyo, Japan
HN

Hiroshi Nishimasu

Professor
Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo
Meguro-ku, Tokyo, Japan
HU

Hideki Uosaki

Associate Professor
Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan
YH

Yutaka Hanazono

Professor
Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University
Shimotsuke, Tochigi, Japan
ON

Osamu Nureki

Professor
Department of Biological Sciences, Graduate School of Science, The University of Tokyo
Bunkyo Ward, Tokyo, Japan

Presenting Author(s)

Tsukasa Ohmori, MD, PhD (he/him/his)

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

Background: An approach to correct disease-specific mutations is the ideal gene therapy for congenital diseases. Base editing is a novel genome-editing technology that can convert a specific DNA base into another at a targeted genome. Point mutations are the most common type of mutation in hemophilia, and hemophilia B has a particularly high percentage of point mutations, even in severe cases, making it a good target disease for base editing. However, the PAM sequence requirement by Cas9 (NGG for SpCas9) hinders its application in various mutations.

Aims: We aimed to restore a disease mutation of the severe hemophilia B patient using a base-editing approach based on SpCas9-NG, an engineered Cas9 with broad PAM flexibility.

Methods: We generated induced pluripotent stem cells (iPSCs) from a hemophilia B patient (c.947T>C; I316T). We established the HEK293 cells and knock-in mice expressing the patient’s F9 cDNA. Cytidine base editor (C>T) comprising the D10A nickase version of Cas9 (wild type SpCas9 or SpCas9-NG) conjugated with TAID or BE4 was transduced into the cells and the mice by plasmid transfection and adeno-associated virus vector, respectively.

Results: We confirmed the broad PAM flexibility of SpCas9-NG near the F9 mutation of the patient. The base editor fused with SpCas9-NG, but not wild-type SpCas9, induced the C to T conversion at the mutation in the patient-derived iPSCs. HEK293 cells and the knock-in mice expressing the patient’s mutation expressed coagulation factor IX (FIX) but did not produce the FIX antigen in the supernatant or plasma. We found that the gene correction of the mutation by SpCas9-NG-mediated base-editing restored the production of the coagulation factor in HEK293 cells and knock-in mice with the mutation.

Conclusion(s): A base-editing approach using Cas9 with broad PAM flexibility would be an attractive approach to correct the point mutation of genetic diseases, including hemophilia B.