Modular flexibility of dystrophin: Implications for gene therapy of Duchenne muscular dystrophy

Abstract

Attempts to develop gene therapy for Duchenne muscular dystrophy (DMD) have been complicated by the enormous size of the dystrophin gene. We have performed a detailed functional analysis of dystrophin structural domains and show that multiple regions of the protein can be deleted in various combinations to generate highly functional mini- and micro-dystrophins. Studies in transgenic mdx mice, a model for DMD, reveal that a wide variety of functional characteristics of dystrophy are prevented by some of these truncated dystrophins. Muscles expressing the smallest dystrophins are fully protected against damage caused by muscle activity and are not morphologically different from normal muscle. Moreover, injection of adeno-associated viruses carrying micro-dystrophins into dystrophic muscles of immunocompetent mdx mice results in a striking reversal of histopathological features of this disease. These results demonstrate that the dystrophic pathology can be both prevented and reversed by gene therapy using micro-dystrophins.

Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin

“…Importantly, the minidystrophin-expressing myofibers excluded Evans blue dye, indicating a functional preservation of sarcolemmal integrity in these fibers (Figure 2b). These results indicated that integrating lentiviral vectors can transduce dystrophic muscles with a minidystrophin gene, which is both larger and more functional than microdystrophin genes being tested in rAAV vectors, 4 and that expression persists for at least 6 months.…”

“…The minidystrophin encoded by this cDNA (DH2-R19) contains eight spectrin-like repeats and has been shown to be fully functional when analyzed in muscles of transgenic mdx mice. 4 We transduced proliferating mdx myoblasts, a permanent myogenic cell line derived from mdx mice (Hauschka, unpublished data), at an MOI of 100 using L-CMV-DH2-R19 vectors. The transduced myoblasts were expanded in growth medium for several days, and then induced to form myotubes.…”

“…2 We and others have demonstrated that replacement of dystrophin in transgenic mdx mice restores normal expression of the dystrophinglycoprotein complex and prevents skeletal muscle pathology from developing. 3,4 Also, delivery of full-length and some truncated dystrophins to adult skeletal muscles using viral vectors has been shown to reverse partially the dystrophic pathology in mdx limb muscles. 2,[4][5][6] Successful gene and cell therapy for DMD will require sustained dystrophin expression in skeletal and cardiac muscles.…”

“…3,4 Also, delivery of full-length and some truncated dystrophins to adult skeletal muscles using viral vectors has been shown to reverse partially the dystrophic pathology in mdx limb muscles. 2,[4][5][6] Successful gene and cell therapy for DMD will require sustained dystrophin expression in skeletal and cardiac muscles. 2 To date, most efforts toward dystrophin delivery have used adenoviral (Ad) or recombinant adeno-associated viral (rAAV) vectors, neither of which has been shown to integrate into myonuclear genomic DNA.…”

“…7,8 Consequently, while these vectors can efficiently transduce striated muscle, their ability to support transgene expression for more than a few years is unclear. 4,[9][10][11] Lentiviral vectors derived from the human immunodeficiency virus-1 (HIV-1) can integrate into the host genome and achieve long-term transgene expression in a wide variety of dividing and nondividing cells including skeletal muscle. [12][13][14][15] The potential use of this vector system for stable gene transfer to muscle is therefore of great interest, as it could theoretically be used for gene transfer to postmitotic myofibers or to a variety of stem cells with myogenic potential.…”