Primary myoblast-mediated gene transfer: persistent expression of human factor IX in mice

Primary myoblast-mediated gene transfer was tested for its ability to mediate a persistent expression of recombinant human factor IX in SCID mice. Mouse primary myoblasts were transduced with factor IX retroviral vectors, LIXSN, which uses the retroviral long-terminal repeats as the promoter, or dLMMBAIX, which uses muscle creatine kinase enhancer and beta-actin promoter to drive factor IX transcription. In vitro, myoblasts transduced with either LIXSN or dLMMBAIX expressed recombinant human factor IX with full biological activity. Upon implantation of transduced myoblasts into skeletal muscles of SCID mice, a sustained systemic expression of factor IX at a level of 10-30 ng/ml plasma was achieved. This was further supported by the presence of recombinant factor IX protein and mRNA in muscle tissues after 5 months of myoblast implantation. Intramuscular implantation of the transduced myoblasts resulted in a gene transfer which was confined locally to the region of injection, with no dissemination to other organs and tissues including testis. Additionally, basic fibroblast growth factor co-injected with primary myoblasts significantly improved the expression level of recombinant factor IX in vivo. These results demonstrate that primary myoblast-mediated gene therapy for hemophilia B is feasible and safe, and can be optimized by using cytokines or other conditions which augment myoblast survival and fusion with myofibers.