In vitro stability of therapeutically relevant, internally truncated dystrophins

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.

Dose-Escalation Study of Systemically Delivered rAAVrh74.MHCK7.micro-dystrophin in the mdx Mouse Model of Duchenne Muscular Dystrophy

“…Use of hinge 2 has the advantage of maintaining the natural linkage to the R1–3 domain, whereas use of hinge 3 would create a novel junction. Novel juxtapositions of protein domains may promote immune responses 59 or result in protein misfolding, which in turn may either promote degradation 60 , 61 or result in formation of protein inclusions. 34 , 62 …”

Development of Novel Micro-dystrophins with Enhanced Functionality

“… 47 , 48 The juxtaposition of different SRs and hinges that are not adjacent to one another in the full-length protein also affect the tertiary structure, stability, and solubility of μDys. 19 , 42 , 49 , 50 Nonetheless, the carboxy-terminal and most of the SR domains can be removed from dystrophin with only modest reductions of striated muscle performance. 15 , 19 The variable degrees of effectiveness of μDys tested to date suggested that versions with improved function might be designed.…”

Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy

“…There is thus still a need for a fully quantitative and standardized method to accurately quantify dystrophin over a wide range of levels, preferably making use of a representative reference control to allow comparison between clinical studies in pre-treatment versus post-treatment samples. Full-length purified dystrophin protein that can serve as a reference standard is not currently available due to the large size and low expression of the dystrophin protein [ 22 ].…”