Platelet-Targeted Gene Therapy of Hemophilia a Using Hematopoietic Stem Cells Derived from Genome-Edited Induced Pluripotent Stem Cells

Hemophilia A (HA), an X-linked recessive hereditary bleeding disorder, is caused by mutations in the F8 gene, which encodes the coagulation factor VIII (FVIII). Replacement therapy is the mainstay in clinic, but it can also cause inhibitory antibodies and is therefore restricted in wide use. Effective therapy of HA with inhibitors needs to be considered as a matter of priority. Evidence has been obtained that functional FVIII can be ectopically expressed and stored in platelets after transplantation of F8 gene modified-hematopoietic stem cells (HSCs), and the stored-FVIII can be released during the activation of platelets to exert therapeutic effect on HA with inhibitors. This unique strategy, however, requires enough sources of HSCs and a safe and effective way of HSCs modification. Induced pluripotent stem cells (iPSCs) are capable of self-renewing, large-scale expansion and differentiating into almost all types of cells and tissues. These features make iPSCs a promising path in the field of regenerative medicine and cell therapy. The recently developed techniques of genome editing, especially the CRISPR/Cas9 system, provide a powerful tool for gene therapy, owing to significantly reduced risk of insertional mutagenesis as compared to the random integration in retroviral-mediated gene therapy. Hence, we seek to explore the possibility of utilizing iPSCs and CRISPR/Cas9 in the platelet-targeted gene therapy of HA.

Here, we established a HA mouse iPSCs (HA-iPSCs) derived from mouse-tail tip fibroblasts, and verified the pluripotency of these cells. On the other hand, we synthesized a codon optimized B domain-deleted FVIII construct (opBDD-F8) in order to enhance the expression efficiency of FVIII. Indeed, our in vitro expression data showed that opBDD-F8 achieved a nearly 4-fold increase of FVIII level as compared to BDD-F8, either under control of the platelet specific GPαIIb promoter or CMV promoter. This increased FVIII expression was further confirmed by hydrodynamic tail vein injection. Then, we inserted the GPαIIb promoter controlled opBDD-F8 (2bopF8) cassette into the Rosa26 locus of HA-iPSCs via CRISPR/Cas9-mediated homologous recombination, and obtained 2bopF8-modified HA-iPSCs (2bopF8-HAiPSCs). No obvious off-target mutation was found in the clones. To generate functional HSCs for transplantation, we performed an in vivo differentiation strategy. We injected 2bopF8-HAiPSCs with hHoxB4 retrovirus transduction into immunodeficient mice to create teratoma, from which we isolated HSCs. The cells were subsequently transplanted into lethally irradiated HA recipient mice. Hematopoiesis was recovered in the recipients, and normal peripheral blood cell numbers sustained for more than 40 weeks. FVIII was detected in platelets of the recipients, suggesting that the cells were originated from 2bopF8-HAiPSCs and FVIII could be expressed during thrombopoiesis. Bleeding phenotype was corrected in the recipients, demonstrating that FVIII released during platelet activation was functional in ensuring coagulation cascade. Furthermore, we did secondary HSC transplantation by using the recipients as the donors. FVIII activity could be detected in the platelets of the new recipients, and bleeding phenotype was corrected in these animals, indicating that long-term HSCs could be generated from 2bopF8-HAiPSCs. During the whole period of research, the recipients were all in good health without teratoma formation or other sickness. Furthermore, we successfully produced chimeric mice with the 2bopF8-HAiPSCs. FVIII expression was observed in the platelets and HSCs were isolated from the mice. Transplantation of these HSCs into lethally irradiated HA mice reconstituted hematopoiesis and generated platelets with FVIII activity. In these mice, coagulation deficiency was also corrected.

Taken together, our proof of principle studies suggest that the genome-edited 2bopF8-HAiPSCs could differentiate towards HSCs with physiological functions, and the functional platelets could be derived from these HSCs. Meanwhile, FVIII could be expressed in platelets and abrogate bleeding diathesis of HA mice. Thus, iPSC-derived HSCs together with genome editing tools represent a potential route for platelet-targeted gene therapy that is worth further investigation.