A scalable, clinically severe pig model for Duchenne muscular dystrophy.

Michael Stirm,Florian Flenkenthaler,Bachuki Shashikadze,Gerhard Wess,Arne Hinrichs,Sibylle Ziegler,Lina Marie Fonteyne,Sabine Krause,Maggie C Walter,Andreas Lange,Christian Kupatt,Kaspar Matiasek,Peter Bartenstein,Mayuko Kurome,Clara Kaufhold,Georg J Arnold,Andrea Bähr,Hiroshi Nagashima,Christian Mayer,Elisabeth Kemter,Thomas Fröhlich,Valeri Zakhartchenko,Andreas Blutke,Roberto Rizzi,Eckhard Wolf,Sven Reese,Rüdiger Wanke,Magdalena Lindner,Claudia Bearzi,Nikolai Klymiuk,Levin Arne Kobelke,Maila Chirivì ,Ivica Međugorac ,Martin Hrabě De Angelis ,Barbara Keßler

doi:10.1242/dmm.049285

Abstract

ABSTRACTLarge-animal models for Duchenne muscular dystrophy (DMD) are crucial for the evaluation of diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMDΔ52) exhibit molecular, clinical and pathological hallmarks of DMD, but die before sexual maturity and cannot be propagated by breeding. Therefore, we generated female DMD+/− carriers. A single founder animal had 11 litters with 29 DMDY/−, 34 DMD+/− as well as 36 male and 29 female wild-type offspring. Breeding with F1 and F2 DMD+/− carriers resulted in an additional 114 DMDY/− piglets. With intensive neonatal management, the majority survived for 3-4 months, providing statistically relevant cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance to human disease mechanisms. Importantly, DMDY/− pigs displayed progressive myocardial fibrosis and increased expression of connexin-43, associated with significantly reduced left ventricular ejection fraction, at 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability. Our breeding cohort of DMDΔ52 pigs and standardized tissue repositories provide important resources for studying DMD disease mechanisms and for testing novel treatment strategies.

Highlights

Duchenne muscular dystrophy (DMD) is a severe, progressive, muscle-wasting disease caused by mutations in the X-linked gene DMD leading to a lack of dystrophin in muscle
The most widely used large animal model for DMD is the golden retriever muscular dystrophy (GRMD) dog, which has a single base change in intron 6 leading to skipping of exon 7 and the termination of the DMD reading frame
A heterozygous DMDΔ52 mutation was introduced in female primary kidney cells (German Landrace × Swabian-Hall hybrid background) by homologous recombination with a modified bacterial artificial chromosome (BAC) in which DMD exon 52 was replaced by a neomycin resistance cassette (Fig. 1A)

Summary

Introduction

Duchenne muscular dystrophy (DMD) is a severe, progressive, muscle-wasting disease caused by mutations in the X-linked gene DMD (encoding dystrophin) leading to a lack of dystrophin in muscle. The classical mdx mouse with a nonsense mutation in Dmd exon 23 and several other strains modeling specific human mutations are most widely used, but have limitations regarding the resemblance of the human disease phenotype. They do not develop early dilated cardiomyopathy as seen in patients (reviewed in (Yucel et al, 2018)). Generation of large animal numbers, sufficient for relevant statistical group sizes, is limited by long generation intervals due to season breeding and relatively small litter sizes (reviewed in (Kornegay, 2017)) This applies for other dog lines with spontaneous DMD mutations (summarized in (Kornegay, 2017)). Attempts to generate a porcine DMD model by injection of CRISPR/Cas into zygotes were hampered by mosaicism and early death of the offspring (Yu et al, 2016)

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Conclusion