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

Abstract

Duchenne muscular dystrophy (DMD) is the most severe muscular dystrophy. It is caused by the absence of dystrophin in muscle fibers. The autologous transplantation of genetically corrected muscle precursor cells (MPCs) is a possible cure for DMD. A non-viral method of genetic modification was tested in this study. The co-transfection (nucleofection) of a phiC31 integrase and a transgene expressing plasmid in MPCs led to an increased stable expression in vitro. The stable expression of a small transgene (eGFP) in muscle fibers was initially demonstrated following the transplantation of the genetically modified cells. The stable expression of a truncated version of dystrophin as well as the full-length dystrophin fused with eGFP was then demonstrated in MPCs obtained from an mdx mice. The transplantation of these cells led not only to the expression of these fusion proteins in muscle fibers but also to the reconstitution of the dystrophin complex. Human MPCs were also genetically modified with a plasmid coding for the fulllength human dystrophin gene fused with eGFP and transplanted in severe combined immuno deficient mice leading to the expression of eGFP dystrophin in muscle fibers. This work indicates that cell transplantation after correction of MPCs with phiC31 integrase is a possible approach to treat DMD.

Site-Specific Recombination Using PhiC31 Integrase

“…After transplanting one million corrected cells from these two groups into irradiated tibialis anterior muscles of mdx mice, immuno fl uorescence revealed that 3-6 weeks later, 250 muscle fi bers per crosssection were positive for the shortened fusion protein and fewer fi bers were positive for the full-length fusion. The restoration of the dystrophin complex was con fi rmed via co-localization of the dystrophin-associated proteins a -sarcoglycan and b -dystroglycan, which were found to be correctly localized at the membrane of the muscle fi bers (Quenneville et al 2007 ) . Additionally, human muscle precursor cells tranfected with a GFP-expressing donor plasmid and an integrase plasmid were observed to express GFP and grow for at least 35 days in vitro, following 1 week of selection.…”

“…Additionally, human muscle precursor cells tranfected with a GFP-expressing donor plasmid and an integrase plasmid were observed to express GFP and grow for at least 35 days in vitro, following 1 week of selection. These cells were then transplanted into the tibialis anterior muscles of SCID mice, which can tolerate xenografts (Quenneville et al 2007 ) . After 1 month, the muscles were harvested, and the human cells were observed to have fused with mouse muscle fi bers and to express GFP.…”

“…A follow-up study (Quenneville et al 2007 ) examined the ability of engineered muscle precursor cells to form corrected muscle fi bers after transplantation. MD1 cells were co-transfected with an integrase-expressing plasmid and either a shortened or a full-length GFP-dystrophin fusion donor plasmid, both under the control of a muscle-speci fi c promoter (Quenneville et al 2007 ) .…”

“…MD1 cells were co-transfected with an integrase-expressing plasmid and either a shortened or a full-length GFP-dystrophin fusion donor plasmid, both under the control of a muscle-speci fi c promoter (Quenneville et al 2007 ) . After transplanting one million corrected cells from these two groups into irradiated tibialis anterior muscles of mdx mice, immuno fl uorescence revealed that 3-6 weeks later, 250 muscle fi bers per crosssection were positive for the shortened fusion protein and fewer fi bers were positive for the full-length fusion.…”

Designing Non-viral Targeted Integrating Vectors for Genome Engineering in Vertebrates

“…Moreover, several teams reported the ex-vivo correction of genetic human diseases in animal models of liver, skin diseases and Duchenne muscular dystrophy (Olivares et al 2002 ;Ortiz-Urda et al 2003 ;Quenneville et al 2007 ) . In mice, F C31-mediated integration of the gene encoding the human factor IX resulted in sustained expression of the therapeutic protein (Olivares et al 2002 ) .…”