Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

Insights into the role of androgen receptor in human testicular peritubular cells.

Contractile smooth muscle-like peritubular cells build the wall of seminiferous tubules in men. They are crucial for sperm transport and complement the functions of Sertoli cells by secreting factors, including glial cell line-derived neurotrophic factor. Previous studies revealed that they also secrete the chemokine C-X-C motif chemokine ligand 12 (CXCL12), which has known roles in spermatogenesis. Peritubular cells express the androgen receptor (AR), which is retained in isolated human testicular peritubular cells. We aimed to explore AR-regulated functions in human testicular peritubular cells. Bearing in mind that infertile men often have high aromatase activity, which may lower intratesticular androgen concentrations, an animal model for male infertility was studied. These mice display an age-dependent loss in spermatogenesis due to high aromatase activity. Human testicular peritubular cells were exposed to dihydrotestosterone or the antiandrogen flutamide. We studied AR, smooth muscle cell markers, glial cell line-derived neurotrophic factor and 15 secreted factors previously identified, including CXCL12. We used qPCR, Western blotting, ELISA or selected reaction monitoring (SRM). In the animal model for male infertility, we employed qPCR and immunohistochemistry. Dihydrotestosterone increased AR and flutamide prevented these actions. The smooth muscle cell markers calponin and smooth muscle actin were likewise increased, while cell size or cellular proliferation was not changed. Dihydrotestosterone did not increase glial cell line-derived neurotrophic factor or CXCL12 secretion but increased levels of serine proteinase inhibitor (SERPIN) E1. The animal model for male infertility with high aromatase activity showed reduced numbers of AR-immunoreactive testicular peritubular cells, suggesting that altered androgen and/or oestrogen levels could influence AR-mediated responses in peritubular cells. Androgens act on human testicular peritubular cells to enhance AR levels, their contractile phenotype and to modulate the secretion of some secreted factors. This study suggests that some aspects of human peritubular cell functions are regulated by androgens.

Characterization of a non-human primate model for the study of testicular peritubular cells-comparison with human testicular peritubular cells.

STUDY QUESTION: Are monkey testicular peritubular cells (MKTPCs) from the common marmoset monkey (Callithrix jacchus) a suitable translational model for the study of human testicular peritubular cells (HTPCs)? SUMMARY ANSWER: MKTPCs can be isolated and propagated in vitro, retain characteristic markers for testicular peritubular cells and their proteome strongly (correlation coefficient of 0.78) overlaps with the proteome of HTPCs. WHAT IS KNOWN ALREADY: Smooth-muscle-like peritubular cells form the wall of seminiferous tubules, transport sperm, are immunologically active, secrete a plethora of factors and may contribute to the spermatogonial stem cell niche. Mechanistic studies are hampered by heterogeneity of human samples. STUDY DESIGN, SIZE, DURATION: We established a culture method for MKTPCs and characterized these cells from six young adult animals (2-3 years). To examine whether they qualify as a translational model we also examined HTPCs from seven men and compared the proteomes of both groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used explant cultures to obtain MKTPCs, which express smooth muscle markers (calponin (CNN1), smooth muscle actin (ACTA2)), lack FSH-receptors (FSHR) and LH-receptors (LHCGR), but possess androgen receptors (AR). MKTPCs can be passaged at least up to eight times, without discernable phenotypic changes. Mass-spectrometry-based analyses of the MKTPC and HTPC proteomes were performed. MAIN RESULTS AND THE ROLE OF CHANCE: We established a method for isolation and cultivation of MKTPCs, and provide a comprehensive analysis of their protein repertoire. The results let us conclude that MKTPCs are suitable as a non-human primate model to study peritubular cell functions. LARGE SCALE DATA: List of identified proteins in MKTPCs by liquid chromatography-tandem mass spectrometry is accessible at the ProteomeXchange (identifier PXD009394). LIMITATIONS, REASON FOR CAUTION: This is an in vitro cellular non-human primate model used to provide a window into the role of these cells in the human testis. WIDER IMPLICATIONS OF THE FINDINGS: Previous studies with HTPCs from patients revealed a degree of heterogeneity, possibly due to age, lifestyle and medical history of the individual human donors. We anticipate that the new translational model, derived from young healthy non-human primates, may allow us to circumvent these issues and may lead to a better understanding of the role of peritubular cells. STUDY FUNDING AND COMPETION OF INTEREST(S): This work was supported by grants from the Deutsche Forschungsgemeinschaft (MA 1080/27-1; AR 362/9-1; BE 2296/8-1). The authors declare no competing financial interests.

Human testicular peritubular cells secrete pigment epithelium-derived factor (PEDF), which may be responsible for the avascularity of the seminiferous tubules.

Male fertility depends on spermatogenesis, which takes place in the seminiferous tubules of the testis. This compartment is devoid of blood vessels, which are however found in the wall of the seminiferous tubules. Our proteomic study using cultured human testicular peritubular cells (HTPCs) i.e. the cells, which form this wall, revealed that they constitutively secrete pigment epithelium-derived factor, PEDF, which is known to exert anti-angiogenic actions. Immunohistochemistry supports its presence in vivo, in the human tubular wall. Co-culture studies and analysis of cell migration patterns showed that human endothelial cells (HUVECs) are repulsed by HTPCs. The factor involved is likely PEDF, as a PEDF-antiserum blocked the repulsing action. Thus testicular peritubular cells, via PEDF, may prevent vascularization of human seminiferous tubules. Dihydrotestosterone (DHT) increased PEDF (qPCR) in HTPCs, however PEDF expression in the testis of a non-human primate occurs before puberty. Thus PEDF could be involved in the establishment of the avascular nature of seminiferous tubules and after puberty androgens may further reinforce this feature. Testicular microvessels and blood flow are known to contribute to the spermatogonial stem cell niche. Hence HTPCs via control of testicular microvessels may contribute to the regulation of spermatogonial stem cells, as well.