Human umbilical cord mesenchymal stem cell-based gene therapy for hemophilia B using scAAV-DJ/8-LP1-hFIXco transduction.

ABSTRACT

BACKGROUND: Hemophilia B is an X-linked bleeding disorder caused by a mutation in the gene responsible for encoding coagulation factor IX (FIX). Gene therapy offers promising potential for curing this disease. However, the current method of relatively high dosage of virus injection carries inherent risks. The purpose of this study was to introduce a novel scAAV-DJ/8-LP1-hFIXco vector transduced human umbilical cord blood derived mesenchymal stem cells (HUCMSCs) as an alternative cell-based gene therapy to conventional gene therapy for Hemophilia B. METHODS: The LP1-hFIXco gene structure was designed by us through searching the literature from NCBI and the scAAV-DJ/8-LP1-hFIXco vector was constructed by a commercial company. The HUCMSCs were cultivated in routine approach and transduced with scAAV-DJ/8-LP1-hFIXco vector. The human FIX activation system was employed for detection of hFIXco activity. The RNA and protein expression levels of the hFIXco were evaluated using PCR and western blot techniques. In animal studies, both NSG and F9-KO mice were used for the experiment, in which clotting time was utilized as a parameter for bleeding assessment. The immunohistochemical analysis was used to assess the distribution of HUCMSCs in mouse tissue sections. The safety for tumorigenicity of this cell-based gene therapy was evaluated by pathological observation after hematoxylin-eosin staining. RESULTS: The transduction of HUCMSCs with the scAAV-DJ/8-LP1-hFIXco vector results in consistent and sustainable secretion of human FIXco during 5 months period both in vitro and in mouse model. The secretion level (hFIXco activity 97.1 ± 2.3% at day 7 to 48.8 ± 4.5% at 5 months) was comparable to that observed following intravenous injection with a high dose of the viral vector (hFIXco activity 95.2 ± 2.2% to 40.8 ± 4.3%). After a 5-month observation period, no clonal expansions of the transduced cells in tissues were observed in any of the mice studied. CONCLUSIONS: We have discovered a novel and safer HUCMSCs mediated approach potentially effective for gene therapy in hemophilia B.