Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin

Abstract

Gene therapy for Duchenne muscular dystrophy (DMD) will require sustained expression of therapeutic dystrophins in striated muscles. Lentiviral vectors have a relatively large transgene carrying capacity and can integrate into nondividing cells. We therefore explored the use of lentiviral vectors for transferring genes into mouse skeletal muscle cells. These vectors successfully transferred a minidystrophin expression cassette into mdx muscles, and minidystrophin expression persisted and prevented subsequent muscle fiber degeneration for at least 6 months. However, only low to moderate levels of skeletal muscle transduction could be obtained by intramuscular injection of the highest currently available lentiviral doses. Using cultured cells, the lentiviral vectors effectively transduced proliferating and terminally differentiated muscle cells, indicating that cell cycling is not essential for transduction of myogenic cells. We further showed that lentiviral vectors efficiently transduced both primary myoblasts and multipotent adult progenitor cells (MAPCs) in vitro, and the cells persistently expressed transgenes without any obvious toxicity. When mdx primary myoblasts were genetically modified with minidystrophin vectors and transplanted into mdx skeletal muscles, significant numbers of dystrophin-expressing myofibers formed. Finally, we showed that a short, highly active CK6 regulatory cassette directed muscle-specific activity in the context of the lentiviral vectors. The ability of lentiviral vectors to transduce myogenic progenitors using a minidystrophin cassette regulated by a muscle-specific promoter suggests that this system could be useful for ex vivo gene therapy of muscular dystrophy.

Full‐length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells

“…Our lentivirus construction recapitulates the transduction efficiency of previously tested and published lentivirus vectors [38,39]. We performed FACS analysis to determine the expression of GFP in infected CD133+ normal blood derived cells and we showed a percentage of 60.9% of GFP positive cells demonstrating a good efficiency of transduction as previously described [40,41] (Fig. 3B).…”

“…The PCR product woas digested with NheI and subcloned into pRRL-cPPT-hPGK-eGFP-WPRE vector. The transduction efficiency of lentiviral vectors containing eGFP and the dysferlin cassette was tested using the same multiplicity of infection as previously described [40,41]. Both lentiviral vectors showed the same transduction efficiency.…”

Duchenne Muscular Dystrophy: Therapeutic Approaches to Restore Dystrophin

“…Lentiviral vectors have been used to treat mdx mice locally restoring dystrophin expression at different efficiencies (Kobinger et al, 2003;Li et al, 2005). Kimura and colleagues showed that a lentivirus encoding μDys intramuscularly injected into 2 weeks old mdx 4cv mice could restore dystrophin in up to 400-1200 fibers in the tibialis anterior muscle.…”

Stem Cell Based Therapy for Muscular Dystrophies: Cell Types and Environmental Factors Influencing Their Efficacy

“…However key questions that need to be resolved before this approach could be used in DMD include the optimal vector configuration and the safety profile of the gene delivery methodology. Lentiviral vectors efficiently infect quiescent cells, including stem cells (S. Li et al, 2005) and give long-term, heritable, gene expression because they integrate into the host genome. Drawbacks with lentiviral vectors include possible gene silencing, or mutagenesis (Wilson & Cichutek, 2009), due to the site at which the virus inserts into the host genome.…”

“…A lentiviral vector has been used to insert a 6.8 kb dystrophin mini gene (S. Li et al, 2005), to give rise to a shorter dystrophin protein in regenerated muscle fibers. While these engineered mini-dystrophins appear to retain most of the functional properties of full-length dystrophin, they nevertheless miss important domains, such as the nitric oxide synthase anchoring domain (Lai et al, 2009).…”