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Poster Networking

Inherited Bleeding Disorders and Inhibitors

PB0561 - Developing cell-based genome editing approach for antithrombin deficiency: a therapeutic prospect

Monday, June 24, 2024
13:45 – 14:45 ICT
Room: Exhibition Hall
Background: Inherited deficiency of antithrombin (AT) is the most severe thrombophilia with 5 to 50 fold increased risk for venous thromboembolism. In some cases, severe clinical events occur despite anticoagulant treatment. Until now, there is no available treatment targeting the disease-causing mutation.

Aims: To investigate the therapeutic potential of gene editing combined with stem cells and hepatic organoid (HO) technology for correcting the SERPINC1 mutation and restore AT production in cells from an AT deficient patient.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of a patient with AT deficiency carrying the heterozygous missense mutation c.350 C>A. The PBMCs were reprogrammed into induced pluripotent stem cells (iPSCs) using a non-integrative sendai virus. Pluripotency of the patient-derived iPSCs was assessed by trilineage differentiation. CRISPR/ Cas9 was used to correct the genetic defect in these iPSCs which were subjected to clonal selection, and the correction of the mutation was confirmed by Sanger sequencing. The edited iPSCs were differentiated into HO using a small molecule based protocol. An ELISA approach was employed to assess AT secretion in both the mutant and gene-corrected HOs.

Results: The patient PBMCs were successfully reprogrammed into stem cells capable of differentiating into all three germ layers (endoderm, ectoderm, and mesoderm within 3 months, confirming their pluripotency). Utilizing CRISPR/Cas9 technology, the missense mutation in the AT loci of the patient-derived iPSCs was reverted to a wild-type genotype. The HOs derived from the genome-edited iPSCs exhibited a 10-fold increase in AT protein secretion as compared to the iPSC-HOs carrying the mutation in AT loci (309.6 ng/ mL and 25 ng/mL respectively).

Conclusion(s): Correction of a heterozygous missense mutation in SERPINC1 restored AT production in patient-derived HOs, thus demonstrating the potential for utilizing ex vivo gene editing and autologous cell therapy as novel treatment methods in AT deficiency.