An induced pluripotent stem cell-mediated and integration-free factor VIII expression system

Abstract

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance and the ability to carry large gene inserts. Induced pluripotent stem (iPS) cells also have a great potential for gene therapy, which can be generated from an individual's own tissues and contribute to any tissues when reintroduced. A Sendai virus (SeV) vector with reprogramming factors is a powerful tool for generating iPS cells because of the high infection efficiency without the risk of integration into host chromosomes. In this study, we developed an iPS cell-mediated and integration-free coagulation factor VIII (FVIII) expression system using non-integrating SeV- and HAC-vectors. Multiple human FVIII genes, which were under the control of the megakaryocyte-specific platelet factor-4 (PF4) promoter for development of a treatment for hemophilia A, were inserted into a HAC vector (PF4-FVIII-HAC). The PF4-FVIII-HAC was introduced into SeV vector-mediated iPS cells derived from a mouse model of hemophilia A. After in vitro differentiation of iPS cells with the PF4-FVIII-HAC into megakaryocytes/platelets, the PF4-FVIII-HAC resulted in expression of FVIII. This study has developed the iPS cell-mediated PF4-driven FVIII expression system using two non-integrating vectors; therefore, this system may be a promising tool for safer gene- and cell-therapy of hemophilia A.

Gene therapy for hemophilia

“…HACs are maintained separately from the host genome (minimizing the risk of insertional mutagenesis), persist through cell divisions due to their ability to bind centrosomal proteins, and allow for delivery of large constructs that can mimic physiological gene regulation (347-350). Megakaryocytes/platelets derived from iPS cells have been generated that produce F.VIII following transduction with a HAC, though their in vivo efficacy has not yet been demonstrated (351). …”

Research progress of hereditary blood diseases

“…另 外, 近年来研究者还通过采用基因编辑技术原位修复 致病基因的点突变 [50] , 或将目的基因敲入到指定的表 达水平较高的基因位点(如血清白蛋白基因位点), 异 位表达凝血因子基因 [51] [52] 及Kim研究组 [53] 分别采用TALEN及CRISPR-Cas9 技术成功原位修复父母来源iPS中FⅧ因子22号内含 子的倒位. Yakura等人 [54] http://engine.scichina.com/doi/10.1360/N052017-00271…”

Pluripotent Stem Cells and Gene Therapy

“…A similar ZFN gene-editing system was further applied to correct SCD with the encouraging result that patient-specific hiPSCs could be produced using Cre-mediated excisable transposons. 115 PF4-FVIII-HAC expressing iPSCs were then differentiated into platelets that appropriately expressed Factor VIII. 109 Several studies have been conducted that suggest iPSCs can be used in hemophilia patients with the potential for use in future therapies.…”