Jonuschies, J; (2012) Characterisation of Lentiviral Transgene Expression in Muscle Precursor Cells: Towards a Potential Therapy for Duchenne Muscular Dystrophy. Doctoral thesis , UCL (University College London).

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Abstract

Duchenne Muscular Dystrophy is an X-linked genetic disorder characterised by progressive muscle degeneration due to the absence of functional dystrophin protein. Damaged muscle fibres are initially regenerated by satellite cells, the principal muscle-resident stem cells, which give rise to committed progenitor cells that differentiate and fuse with the damaged muscle fibre to introduce new functional myonuclei. Satellite cells also self-renew to replenish the stem cell pool. Autologous transplantation of genetically-corrected satellite cells presents an attractive strategy to introduce a functional dystrophin copy into dystrophic muscles and to replenish the stem cell pool with corrected satellite cells for a long-term therapeutic benefit. Lentiviral vectors are suitable gene transfer vectors that integrate their genome into the host chromosome and mediate stable transgene expression in dividing and non-dividing cells. Long-term gene expression can be hampered by promoter inactivation and undesirable position effects. Tissue-specific promoters have been shown to reduce the risk of transcriptional silencing and to increase the overall biosafety of transgene expression. In this study, I investigated the potential of integrating lentiviral vectors to efficiently infect quiescent satellite cells in order to confer stable expression of GFP in vitro and in vivo. The effect of viral transduction and GFP overexpression on stem cell properties was assessed. The performance of tissue-specific promoters was directly compared with the strong viral promoter of the spleen focus-forming virus and revealed the desmin promoter as an attractive non-viral alternative to confer stable, high-level, muscle-specific expression in myoblasts and myofibres. An ubiquitously-acting chromatin opening element (UCOE) has been reported to negate position effects in hematopoeitic cells. Here, the UCOE failed to prevent promoter down-regulation and did not significantly increase transgene expression when it was combined with the desmin promoter. In summary, this work provides useful information on suitable promoters to achieve stable transgene expression in the myogenic linage.

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