Steinbrecher, Ricarda Anette; (1996) Characterization of genomic myosin heavy chain gene isoforms and their 5' regulatory elements in rabbit: applications for gene therapy. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

An effective gene therapy approach by direct gene injection into muscle requires a strong expression and preferably inducible vector that is specifically activated in differentiated muscle. Plasmids are known to be taken up by muscle cells after direct gene injection and to be expressed and kept episomally for over a year without posing a risk of random integration into the genome. The use of muscle specific regulatory elements in such plasmids should facilitate the expression of any gene spliced into this construct. Myosin is a major contractile protein of muscle and its expression is regulated in a developmental and fibre type specific fashion. Myosin heavy chain (MyHC) isoforms and myosin light chain (MyLC) isoforms both resemble multigene families and their regulatory elements are highly specific for muscle. To elucidate the complexity and composition of the MyHC gene family and to isolate 5'regulatory elements, 32 genomic MyHC clones were isolated and characterized by restriction mapping, Southern hybridization, sequencing and PCR (polymerase chain reaction). These MyHC clones were classified into 12 potential subgroups and a 5'region of 2865 nucleotides was sequenced and tested for regulatory elements. This thesis reports the use of MyHC and MyLC regulatory elements to drive the expression of exogenous genes, reporter genes as well as the gene for blood clotting factor VIII, in transfected myoblasts. The results of the in vitro studies suggest that this construct and this procedure may be suitable for use in animal models of haemophilia and might thus represent a step towards gene therapy for haemophilia A.

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