In vitro stability of therapeutically relevant, internally truncated dystrophins

Jackie L Mccourt,Joseph J Belanto,Katrina K Rhett,James M Ervasti,Dana M Talsness,Michele A Jaeger

doi:10.1186/s13395-015-0040-z

Jackie L Mccourt, Joseph J Belanto + Show 4 more

Open Access

https://doi.org/10.1186/s13395-015-0040-z

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Journal: Skeletal Muscle	Publication Date: Apr 28, 2015
Citations: 67	License type: CC BY 4.0

Affiliation: University of Minnesota

Abstract

BackgroundThe X-linked recessive disease Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding the protein dystrophin. Despite its large size, dystrophin is a highly stable protein, demonstrating cooperative unfolding during thermal denaturation as monitored by circular dichroism spectroscopy. In contrast, internal sequence deletions have been associated with a loss of the cooperative unfolding and cause in vitro protein aggregation. Several emerging therapy options for DMD utilize internally deleted micro-dystrophins and multi-exon-skipped dystrophins that produce partially functional proteins, but the stability of such internally truncated proteins has not been investigated.MethodsIn this study, we analyzed the in vitro stability of human dystrophin constructs skipped around exon 45 or exon 51, several dystrophin gene therapy constructs, as well as human full-length and micro-utrophin. Constructs were expressed in insect cells using the baculovirus system, purified by affinity chromatography, and analyzed by high-speed sedimentation, circular dichroism spectroscopy, and differential scanning fluorimetry.ResultsOur results reveal that not all gene therapy constructs display stabilities consistent with full-length human dystrophin. However, all dystrophins skipped in-frame around exon 45 or exon 51 show stability profiles congruent with intact human dystrophin. Similar to previous studies of mouse proteins, full-length human utrophin also displays stability similar to human dystrophin and does not appear to be affected by a large internal deletion.ConclusionsOur results suggest that the in vitro stability of human dystrophin is less sensitive to smaller deletions at natural exon boundaries than larger, more complex deletions present in some gene therapy constructs.

Highlights

The X-linked recessive disease Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding the protein dystrophin
The gene therapy constructs μH2 human dystrophin and μH3 hDys contain spectrin-like repeats (SLRs) 1 to 3 and 24 with hinge 2 or hinge 3, respectively
The Δ3990 hDys construct corresponds to the AAVdelivered micro-dystrophin used in a clinical trial that reported minimal recombinant dystrophin expression associated with a strong immune response to dystrophin [20,37]

Summary

Introduction

The X-linked recessive disease Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding the protein dystrophin. The X-linked disease Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding the protein dystrophin [1]. Mutations causing this disease are variable with 65% of DMD patients harboring deletions which span exons, 5% to 15% having duplications, and the remaining populations having either point mutations or deep intronic deletions [2]. Becker muscular dystrophy (BMD) is a milder allelic form of dystrophy typically caused by in-frame gene deletions that maintain reading frame but presumably cause disease through diminished abundance or functionality [3]. The observation that milder BMD patients harbor deletions in the central rod domain suggests that dystrophin can tolerate such deletions to some degree and that the central rod domain is less critical to the function of dystrophin

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