Restoration of Functional Full-Length Dystrophin After Intramuscular Transplantation of Foamy Virus-Transduced Myoblasts.

Stem cell therapy is a promising strategy to treat muscle diseases such as Duchenne muscular dystrophy (DMD). To avoid immune rejection of donor cells or donor-derived muscle, autologous cells, which have been genetically modified to express dystrophin, are preferable to cells derived from healthy donors. Restoration of full-length dystrophin (FL-dys) using viral vectors is extremely challenging, due to the limited packaging capacity of the vectors, but we have recently shown that either a foamy viral or lentiviral vector is able to package FL-dys open-reading frame and transduce myoblasts derived from a DMD patient. Differentiated myotubes derived from these transduced cells produced FL-dys. Here, we transplanted the foamy viral dystrophin-corrected DMD myoblasts intramuscularly into mdx nude mice, and showed that the transduced cells contributed to muscle regeneration, expressing FL-dys in nearly all the muscle fibers of donor origin. Furthermore, we showed that the restored FL-dys recruited members of the dystrophin-associated protein complex and neuronal nitric oxide synthase within donor-derived muscle fibers, evidence that the restored dystrophin protein is functional. Dystrophin-expressing donor-derived muscle fibers expressed lower levels of utrophin than host muscle fibers, providing additional evidence of functional improvement of donor-derived myofibers. This is the first in vivo evidence that foamy virus vector-transduced DMD myoblasts can contribute to muscle regeneration and mediate functional dystrophin restoration following their intramuscular transplantation, representing a promising therapeutic strategy for individual small muscles in DMD.

Delivery of large transgene cassettes by foamy virus vector.

Viral vectors are effective tools in gene therapy, but their limited packaging capacity can be restrictive. Larger clinically-relevant vectors are needed. Foamy viruses have the largest genomes among mammalian retroviruses and their vectors have shown potential for gene therapy in preclinical studies. However, the effect of vector genome size on titre has not been determined. We inserted increasing lengths of the dystrophin open reading frame in a foamy virus vector and quantified packaged vector RNA and integrated DNA. For both measures, a semi-logarithmic reduction in titre was observed as genome size increased. Concentrated titres were reduced 100-fold to approximately 106 transducing units per ml when vector genomes harboured a 12 kb insert, approximately twice that reported for lentivirus vectors in a comparable study. This potential was applied by optimising foamy virus vectors carrying the full-length dystrophin open-reading frame for transduction of human muscle derived cells. Full-length dystrophin protein was expressed and transduced cells remained able to form myotubes in vitro. Foamy virus vectors are well-suited for stable delivery of large transgene cassettes and warrant further investigation for development as a therapy for Duchenne or Becker muscular dystrophy.

Rapid and Efficient Stable Gene Transfer to Mesenchymal Stromal Cells Using a Modified Foamy Virus Vector.

Mesenchymal stromal cells (MSCs) hold great promise for regenerative medicine. Stable ex vivo gene transfer to MSCs could improve the outcome and scope of MSC therapy, but current vectors require multiple rounds of transduction, involve genotoxic viral promoters and/or the addition of cytotoxic cationic polymers in order to achieve efficient transduction. We describe a self-inactivating foamy virus vector (FVV), incorporating the simian macaque foamy virus envelope and using physiological promoters, which efficiently transduces murine MSCs (mMSCs) in a single-round. High and sustained expression of the transgene, whether GFP or the lysosomal enzyme, arylsulphatase A (ARSA), was achieved. Defining MSC characteristics (surface marker expression and differentiation potential), as well as long-term engraftment and distribution in the murine brain following intracerebroventricular delivery, are unaffected by FVV transduction. Similarly, greater than 95% of human MSCs (hMSCs) were stably transduced using the same vector, facilitating human application. This work describes the best stable gene transfer vector available for mMSCs and hMSCs.