Fkrp rescue in blood-derived CD133+ cells isolated from patients affected by congenital muscular dystrophies

Dystroglycanopathies are a group of heterogeneous muscular dystrophies caused by mutations occurring in 6 genes (POMT1, POMT2, POMGnT1, FKRP, Fukutin and LARGE) involved in the glycosylation of α-dystroglycan (α-DG). Actually there are no effective therapies for these disorders. One proposed approach could be the development of a combined gene and cell therapy. For this purpose, we selected patients affected by mutations in the coding sequence of fukutin related protein gene (FKRP). We firstly isolated blood-derived CD133+ cells from healthy and dystrophic patients and demonstrated their self-renewal capacity. HEK cells were transduced with a lentiviral vector coding for the FKRP gene (pLenti-CAG(FKRP)-Rsv(GFP-puro). A pLenti-CAG-Rsv(GFP-puro) was used as control. To evaluate the lentiviral infectious efficiency, we transduced HEK cells with three different increasing MOI: 1, 5, 10; HEK cells showed a high viability after lentiviral infection (more than 80%). The efficiency of transduction increased from 55% (MOI 1), to 88% (MOI 5) until a maximum of 92% (MOI 10). We thus selected MOI 10 for all future experiments performed on blood-derived CD133+ cells. Green fluorescent protein (GFP) expression was used to define the infectious efficacy by cytofluorimetric analysis. Transduced cells maintained proliferation capacity and > 90% vitality. q-RT-PCR and WB analysis confirmed the expression of FKRP in engineered CD133+ cells isolated from dystrophic patients. Engineered human blood-derived CD133+ cells were induced to differentiate in myotubes when co-cultured in the presence of a feeder layer of mouse myogenic cells. Q-RT-PCR and WB analysis confirmed the expression of early myogenic markers (PAX7, MYF5, M-cadherin and MRF4). Data demonstrate that blood-derived CD133+ cells can be easily isolated with no invasive procedure from FKRP mutated patients and manipulated in vitro. Therefore genetically FKRP engineered cells can be considered as a tool for future investigations in dystroglycanopathies.