Nucleofection of muscle-derived stem cells and myoblasts with ϕC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts

Abstract

Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.

Dystrophin expression in host muscle following transplantation of muscle precursor cells modified with the phiC31 integrase

“…15,22 In our previous experiments, these methods were shown to be very efficient for the MPC transfection and stable gene expression in vitro. 11 The present work was carried out to verify whether the gene transfected with this method could be expressed in vivo.…”

“…As very low-transfection levels were observed in the preliminary experiments of nucleofection with the fulllength dystrophin transgene, 11 intermediate experiments using a truncated version of the dystrophin cDNA were carried out. This so-called MDys was fused with eGFP 18 and an attB sequence was also added in the plasmid to produce the p-attB-CMV-eGFP-MDys.…”

“…The plasmid pattB-CMV-eGFP-Dys has been described previously. 11 The pattB-MCK-eGFP-Dys was produced using the same restriction sites (NdeI and AgeI) as described for pattB-MCK-eGFP-MDys. Figure 1 shows most of the constructs.…”

“…9,10 Recently, the Amaxa Corporation developed the nucleofection method, which allowed us to deliver not only eGFP but also the full-length dystrophin fused with eGFP into human primary MPCs. 11 Stable expressions of these transgenes were then obtained with the phiC31 integrase system. In this system, sequences called 'attB' and 'attP' are recognized and recombined by the integrase.…”

Meganucleases can restore the reading frame of a mutated dystrophin

“…As the PCR analysis indicated the presence of the expected amplicon size, the nucleofection of infected human myoblasts was made as previously described. 43 In brief, 5 mg of plasmid MGNs RAG1 or I-SceI was introduced by nucleofection within the infected human myoblasts containing the corresponding specific target. Nucleofector solution adult (NHDF-adult) has been used and the nucleofector apparatus (AMAXA Inc., Amaxa Nucleofector System, Lonza Walkerrsville Inc., Walkersville, MD, USA) was set to program P-022 according to the Amaxa protocol.…”