Gene Therapy Strategies for Duchenne Muscular Dystrophy Utilizing Recombinant Adeno-associated Virus Vectors  Michael J. Blankinship, Paul Gregorevic, Jeffrey S. Chamberlain  Molecular Therapy  Volume 13, Issue 2, Pages 241-249 (February 2006) DOI: 10.1016/j.ymthe.2005.11.001 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Functional domains of dystrophin and structural features of mini- and microdystrophins. Top: The full-length, “muscle-specific” isoform of dystrophin. The protein begins with actin-binding domain 1 (ABD1), which contains two calponin homology (CH) domains that form a hydrophobic interaction with γ-actin filaments. The central domain is composed of four hinges (H) and 24 spectrin-like repeats (R). These repeats fold into triple-helical coiled-coil domains forming a flexible and elastic rod domain. Unshaded repeats are basically charged, and repeats 11–17 form an internal actin binding domain (ABD2) that electrostatically interacts with actin. The dystroglycan-binding domain (DgBD) is composed of a WW domain, a cysteine-rich (CR) domain containing two EF hand-like structures (EF1, EF2), and a zinc-finger “ZZ” domain. The C-terminal (CT) domain binds members of the syntrophin and dystrobrevin protein families at the indicated sites (syn1, syn2, and cc1, cc2, respectively). Portions of the CT domain are absent from some dystrophin isoforms due to alternative splicing of the indicated exons. See [4] for detailed references to the original studies. Middle: Structure of a microdystrophin lacking spectrin-like repeats 4–23, as well as hinge 3 and the CT domain (ΔR4–R23/ΔCT). This construct is highly functional when delivered systemically to mdx muscles using rAAV [35]. Bottom: Structure of a fully functional minidystrophin that lacks 16 of the 24 spectrin-like repeats (R4–19) as well as hinge 2. This construct is also discussed in the legend to Fig. 3 [38] and is fully functional in muscles of transgenic mdx mice [3]. Molecular Therapy 2006 13, 241-249DOI: (10.1016/j.ymthe.2005.11.001) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 The dystrophin–glycoprotein complex in (A) normal and (B) mdx muscles expressing microdystrophins. Indicated in (A) are portions of dystrophin missing from the ΔR4–R23/ΔCT construct (the region between and including R4–R23 and the CT domain). The CT domain binds to various syntrophin (Syn) and dystrobrevin (Dbn) proteins, and syntrophin binds nNOS. In the absence of the CT domain, Dbn remains associated with the DGC by an unclear mechanism, possibly by binding the sarcoglycans (Sg) (B). Dbn also binds syntrophin, such that this latter protein is properly expressed in a complex with microdystrophin, but nNOS is not. Dg, dystroglycan; NT, actin binding domain 1; W, WW domain; CR, cysteine-rich domain; Spn, sarcospan. Molecular Therapy 2006 13, 241-249DOI: (10.1016/j.ymthe.2005.11.001) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Dual-vector strategy to generate proteins too large to be encoded on a single AAV genome. The example shows how a minidystrophin was generated by coadministration of two separate rAAV vectors [38]. The two vectors encoded either the 5′ or the 3′ half of the desired expression cassette (top). The 5′ cassette ends with the 5′ end of a natural dystrophin intron, located at its natural exon/intron boundary. The 3′ cassette is preceded by the 3′ end of the same intron and joins the dystrophin cDNA at its natural exon/intron boundary. After coadministration in vivo, the two vectors form concatemers (middle), and the intron is spliced around the ITRs, forming a mRNA that encodes a larger, fully functional minidystrophin (ΔH2-R19 [3]). Molecular Therapy 2006 13, 241-249DOI: (10.1016/j.ymthe.2005.11.001) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Whole-body microdystrophin gene transfer to the musculature using rAAV6. 1 × 1012 vg of rAAV6/CMV-μDys (ΔrR4–R23/ΔCT—see Fig. 1) was injected into the tail vein of an 8-week-old mdx mouse, and tissues were analyzed 8 weeks later. Vector was administered in a 300-μl bolus injection in physiological Ringer's solution containing 0.008% mouse serum albumin and 2 IU sodium heparin. VEGF was not used in this study. All striated muscles were found to express wild-type levels of the human microdystrophin in essentially every myofiber or myocyte. Shown are representative sections of the heart, quadriceps, and intercostal muscles immunostained with a rabbit polyclonal antibody against the N-terminal domain of dystrophin. Top row, muscles from wild-type mice; middle row, muscles from mdx mice; bottom row, muscles from mdx mice injected with rAAV6/CMV-μDys. Molecular Therapy 2006 13, 241-249DOI: (10.1016/j.ymthe.2005.11.001) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Transcript rescue using rAAV vectors encoding antisense RNA sequences. (A) A rAAV vector was generated that carries a modified U7 snRNA gene that was retargeted to spliceosomes and that contains antisense sequences directed against the intron 23 splice donor and the intron 22 branch point that flank murine dystrophin exon 23 [44]. (B) Following delivery to mdx muscles, the vector generates U7 snRNAs that anneal with the mutant dystrophin pre-mRNA, blocking splicing of exon 23 [black box in (C)], thus excluding the mutant sequences from the final, processed mRNA. This processed mRNA now lacks the premature stop codon carried by exon 23 in mdx mice and leads to production of an essentially full-length dystrophin (D). Molecular Therapy 2006 13, 241-249DOI: (10.1016/j.ymthe.2005.11.001) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions