OC 02.2 - Knock-in genome editing therapy with a single AAV vector harboring an engineered AsCas12f for hemophilia B in mice

Saturday, June 22, 2024

13:15 – 13:30 ICT

Room: Ballroom B2

Presenting Author(s)

Tomoki Togashi, Master

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

Co-Author(s)

TH

Tomohiro Hino

Graduate student
Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine
Kyoto, Kyoto, Japan
SO

Satoshi N. Omura

Graduate student
Department of Biological Sciences, Graduate School of Science, The University of Tokyo
Tokyo, Tokyo, Japan
RN

Ryoya Nakagawa

Graduate student
Department of Biological Sciences, Graduate School of Science, The University of Tokyo
Tokyo, Tokyo, Japan
AH

Atsushi Hoshino

Associate Professor
Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine
Kyoto, Kyoto, Japan
ON

Osamu Nureki

Professor
Department of Biological Sciences, Graduate School of Science, The University of Tokyo
Tokyo, Tokyo, Japan
Tsukasa Ohmori

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

Background: Genome editing therapies that disrupt a target gene become a reality, while there are still impediments to genome editing therapy to insert target genes into the genome. We developed an engineered AsCas12f (enAsCas12f) that is one-third the size of SpCas9 with comparable genome editing activity by deep mutational scanning and structural analysis (Cell 2023; 186: 4920-4935).

Aims: We aimed to develop a treatment strategy for hemophilia B by gene knock-in with a single adeno-associated virus (AAV) vector harboring the enAsCas12f in mice.

Methods: We created a single AAV8 vector consisting of the enAsCas12f, gRNA, the gRNA recognition site, and a part of the murine albumin locus (mAlb) sequence conjugated with 2A-fused human coagulation factor IX (FIX) Padua cDNA. AAV vector was intraperitoneally administrated into neonatal hemophilia B mice. Plasma FIX activity (FIX:C) and FIX protein expression in liver cells were assessed with a one-stage clotting assay and immunohistochemistry, respectively.

Results: AAV vector-mediated expression of enAsCas12f can efficiently induce double-strand break at intron 13 and 14 of mAlb. The intraperitoneal injection of the AAV8 targeting intron 14 and donor sequence to insert FIX cDNA only by homology-directed repair (HDR) into neonatal hemophilia B mice resulted in a significant increase in plasma FIX:C (106.4 ± 21.7%). The FIX protein expression assessed by immunohistochemistry was identified in 2.4 ± 0.4% of liver cells. The administration of AAV8 vector to insert donor sequence only by non-homologous end joining (NHEJ) at intron 13 significantly enhanced plasma FIX:C (296.4 ± 13.6%) and the ratio of FIX-expressing liver cells (6.6 ± 0.6%), compared with those by HDR.

Conclusion(s): The enAsCas12f allows knock-in genome editing therapy for hemophilia B with a single AAV vector in mice. Knock-in of liver cells by this strategy is more efficient with NHEJ than with HDR, even in neonatal mice.