In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector

Jeong Hyeon Lee, Jeong Pil Han, Dong Woo Song, Geon Seong Lee, Beom Seok Choi, Min Jeong Kim, Yeji Lee, Seokjoong Kim, Hyukjin Lee, Su Cheong Yeom

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Recent therapeutic strategies for hemophilia include long-term therapeutic gene expression using adeno-associated virus (AAV) and rebalancing therapy via the downregulation of anticoagulant pathways. However, these approaches have limitations in immune responses or insufficiency to control acute bleeding. Thus, we developed a therapeutic strategy for hemophilia B by a combined rebalancing and human factor 9 (hF9) gene knockin (KI) using a lipid nanoparticle (LNP) and AAV. Antithrombin (AT; Serpin Family C Member 1 [Serpinc1]) was selected as the target anticoagulation pathway for the gene KI. First, the combined use of LNP-clustered regularly interspaced short palindromic repeats (CRISPR) and AAV donor resulted in 20% insertions or deletions (indels) in Serpinc1 and 67% reduction of blood mouse AT concentration. Second, hF9 coding sequences were integrated into approximately 3% of the target locus. hF9 KI yielded approximately 1,000 ng/mL human factor IX (hFIX) and restored coagulation activity to a normal level. LNP-CRISPR injection caused sustained AT downregulation and hFIX production up to 63 weeks. AT inhibition and hFIX protein-production ability could be maintained by the proliferation of genetically edited hepatocytes in the case of partial hepatectomy. The co-administration of AAV and LNP showed no severe side effects except random integrations. Our results demonstrate hemophilia B therapy by a combination of rebalancing and hF9 KI using LNP and AAV.

Original languageEnglish
Pages (from-to)161-172
Number of pages12
JournalMolecular Therapy - Nucleic Acids
Volume32
DOIs
StatePublished - 13 Jun 2023

Bibliographical note

Publisher Copyright:
© 2023 The Author(s)

Keywords

  • CRISPR-Cas9
  • MT: Oligonucleotides: Therapies and Applications
  • antithrombin
  • hemophilia B
  • in vivo knockin
  • lipid nanoparticle

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