Progress and prospects of gene therapy clinical trials for the muscular dystrophies

Abstract

Clinical trials represent a critical avenue for new treatment development, where early phases (I, I/II) are designed to test safety and effectiveness of new therapeutics or diagnostic indicators. A number of recent advances have spurred renewed optimism toward initiating clinical trials and developing refined therapies for the muscular dystrophies (MD's) and other myogenic disorders. MD's encompass a heterogeneous group of degenerative disorders often characterized by progressive muscle weakness and fragility. Many of these diseases result from mutations in genes encoding proteins of the dystrophin-glycoprotein complex (DGC). The most common and severe form among children is Duchenne muscular dystrophy, caused by mutations in the dystrophin gene, with an average life expectancy around 25 years of age. Another group of MD's referred to as the limb-girdle muscular dystrophies (LGMDs) can affect boys or girls, with different types caused by mutations in different genes. Mutation of the α-sarcoglycan gene, also a DGC component, causes LGMD2D and represents the most common form of LGMD. Early preclinical and clinical trial findings support the feasibility of gene therapy via recombinant adeno-associated viral vectors as a viable treatment approach for many MDs. In this mini-review, we present an overview of recent progress in clinical gene therapy trials of the MD's and touch upon promising preclinical advances.

Figure 1.

Generation of a custom rabbit anti-FKRP C-terminal antibody for detection of rAAV6-mediated muscle-specific expression of FKRP. (A) Schematic representation of the AAV6 vector genome for expression of FKRP in striated muscles. CK8: miniaturized mouse creatine kinase regulatory cassette (–41, 85, 86); Kozak: kozak sequence, ACCATGG; FKRP: FKRP cDNA and pA: SV40 poly-adenylation signal. (B) Transverse micrograph of C57Bl/6 tibialis anterior (TA) muscle post-injection of rAAV6-CK8-FKRP (shown in A). FKRP foci (green, rabbit anti-FKRP) colocalize with golgi-specific antibody (red, GM130) in injected (white arrows) and WT control sections (inset, white arrowheads). (C) Longitudinal micrograph demonstrates FKRP-golgi co-localization throughout the myofiber. Additional FKRP-positive staining, not associated with golgi, was observed in injected muscle (asterisks) and did not colocalize with markers of nuclear membrane (D; Nucleoporin P62) or late endosomal compartment (E; EEA1).

Figure 2.

Dystrophin restoration following rAAV6-mediated delivery of CRISPR/Cas9. (A) rAAV nuclease vector containing a muscle-specific regulatory cassette based on the mouse muscle creatine kinase promoter/enhancer element (CK8) (–, 88, 89), nuclear localization signals (NLS) and S. pyogenes Cas9. (B) The rAAV targeting vector containing the POL III U6 promoter driving each guide RNA (gRNA) and the intervening CMV promoter/enhancer driving a mCherry reporter gene. (C) Schematic representation of relative exon targeting in the mdx^4cv mouse and approximate gRNA positions to generate the exon Δ52–53 Dmd gene. (D) A representative 6-week-old male mdx^4cv mouse having received direct injection of rAAV-dual vectors into the tibialis anterior (TA) muscles. (E) Non-injected mdx^4cv control. TA muscles were analyzed via immunofluorescence using antibodies raised against the C-terminus of dystrophin (green) and nuclei (DAPI, blue).