238 Tailored Pig Model of Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is one of the most common genetic diseases in humans, affecting 1 in 3500 boys. It is characterised by progressive muscle weakness and wasting due to mutations in the dystrophin (DMD) gene resulting in absence of dystrophin protein in skeletal muscle. Although curative treatments are currently not available, genetic and pharmacological approaches are under investigation including early-phase clinical trials. Existing animal models in different species (e.g. mdx mouse, GRMD dog) have been instrumental to understand the pathophysiology of DMD, but have several limitations. Importantly, the causative point mutations (mdx mouse: nonsense mutation; GRMD dog: splice mutation) are different from the most common human mutations (out-of-frame deletion of one or several exons of the DMD gene). We used gene targeting in somatic cells and nuclear transfer to generate a genetically tailored pig model of DMD. A bacterial artificial chromosome (BAC) from the porcine DMD gene was modified by recombineering to replace exon 52, resulting in a frame shift in the transcript. Modified BAC were transfected into male neonatal kidney cells, which were screened by quantitative polymerase chain reaction for replacement of exon 52 in the X-linked DMD gene. Eight of 436 cell clones were successfully targeted and 2 of them were used for nuclear transfer. For each of the cell clones, a pregnancy was established by transfer of cloned embryos into recipient gilts. Four piglets of the first litter were live born and killed within 48 h and tissue samples were processed for histological characterisation. Two piglets of the second litter died during birth due to obstetric complications, whereas the other 2 piglets were delivered by Caesarean section and raised in an artificial feeding system. Their serum creatine kinase (CK) levels were grossly elevated. Although both piglets showed reduced mobility compared with age-matched controls, they were able to move and feed on their own. Immunofluorescence staining of dystrophin was negative in muscle fibres of DMD mutant piglets and the complete absence of dystrophin protein was confirmed by immunoblot analysis. Histological examination of biceps femoris muscle from DMD mutant pigs showed a degenerative myopathy with fibre size variation, rounded fibres, central nuclei, fibrosis and fatty replacement of muscle tissue mimicking the hallmarks of the human disease. In conclusion, we generated the first pig model for a genetic muscle disease. The DMD mutant pig appears to be a bona fide model of the human dystrophy as ascertained by absence of the dystrophin protein, elevated serum CK levels and early degenerative changes on muscle histology. Because deletion of exon 52 is one of the most frequent mutations found in human DMD, the exon 52 mutated DMD pig represents an excellent model for testing targeted genetic treatments. This study was supported by the Bayerische Forschungsstiftung.

Recent advances in Duchenne muscular dystrophy

“…While HFMD represents spontaneous dystrophin deficiency as a result of an extensive promoter deletion, 51 it is not widely used to limited pathological similarity to DMD. 38 In contrast, the exon 52-deleted 238-DMD pig, similar to mdx52, was engineered to assess exon 51 skipping methodologies, 50 and appears to be a bona fide model, as ascertained by absence of dystrophin protein, elevated serum CK levels, and early degenerative changes on muscle histology. 50 Further, porcine models have a number of practical advantages, such as the ability to circumvent numerous issues that currently preclude experimental transition from mdx into larger models (such as transgenic manipulation and breeding considerations), while retaining a similar size and physiology to humans.…”

“…Hypertrophic feline muscular dystrophy (HFMD) 49 and the 238 tailored pig model (238-DMD) 50 represent two large animal models of DMD that substantially differ in their genetic derivation that are suitable candidates for therapeutic assessment. While HFMD represents spontaneous dystrophin deficiency as a result of an extensive promoter deletion, 51 it is not widely used to limited pathological similarity to DMD.…”

Genetically Engineered Pig Models for Human Diseases

“…Some of those described are in the early stages of characterization, and it is not yet known how useful they will be for modeling specific diseases. Other models that are not discussed above, and which are in the very early stages of characterization, include muscular dystrophy (97), psoriasis-like phenotype (98), and osteoporosis (99). As the pig is shown to be a suitable model for diseases for which a good model does not exist currently, additional genetic modifications will continue to be made.…”

39 Factors Influencing the Efficiency of Generating Genetically Engineered Pigs by Nuclear Transfer: Multi-Factorial Analysis of a Large Data Set

“…Of the gene targeting experiments, only mutation of the X-linked dystrophin (DMD) gene in male clones may cause a severe phenotype. In fact, DMD mutant male piglets showed severe muscular dystrophy already at birth, and a proportion died shortly later [42]. For all other target genes, the heterozygous knockout had either no specific phenotype or a phenotype that develops later in life.…”