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Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

Muscles of patients with facioscapulohumeral dystrophy (FSHD) are characterized by sporadic DUX4 expression and oxidative stress which is at least partially induced by DUX4 protein. Nevertheless, targeting oxidative stress with antioxidants has a limited impact on FSHD patients, and the exact role of oxidative stress in the pathology of FSHD, as well as its interplay with the DUX4 expression, remain unclear. Here we set up a screen for genes that are upregulated by DUX4 via oxidative stress with the aim to target these genes rather than the oxidative stress itself. Immortalized human myoblasts expressing DUX4 (MB135-DUX4) have an increased level of reactive oxygen species (ROS) and exhibit differentiation defects which can be reduced by treating the cells with classic (Tempol) or mitochondria-targeted antioxidants (SkQ1). The transcriptome analysis of antioxidant-treated MB135 and MB135-DUX4 myoblasts allowed us to identify 200 genes with expression deregulated by DUX4 but normalized upon antioxidant treatment. Several of these genes, including PITX1, have been already associated with FSHD and/or muscle differentiation. We confirmed that PITX1 was indeed deregulated in MB135-DUX4 cells and primary FSHD myoblasts and revealed a redox component in PITX1 regulation. PITX1 silencing partially reversed the differentiation defects of MB135-DUX4 myoblasts. Our approach can be used to identify and target redox-dependent genes involved in human diseases., Graphical abstract Image 1, Highlights • Double homeobox transcription factor DUX4 misregulates hundreds of genes and induces oxidative stress in human myoblasts. • ROS, notably those of mitochondrial origin, contribute to the differentiation defects in myoblasts expressing DUX4. • A subset of genes is deregulated by DUX4 indirectly, via oxidative stress. • A strategy to identify the genes deregulated by DUX4 via oxidative stress was developed. • PITX1 is deregulated by DUX4 via oxidative stress and can be targeted to improve myogenesis in DUX4-expressing myoblasts.

DUX4, a double homeobox transcription factor, has been mostly studied in facioscapulohumeral dystrophy (FSHD), a pathology linked to a deletion of subtelomeric repeats on chromosome 4q. More recently, however, the gene has been associated with various sarcomas and haematological malignancies. Drugs developed for FSHD could be tested on cancer cells to develop efficient treatment strategies for both pathologies.

Muscular dystrophies are a group of heterogeneous genetic disorders characterized by progressive loss of skeletal muscle mass. Depending on the muscular dystrophy, the muscle weakness varies in degree of severity. The majority of myopathies are due to genetic events leading to a loss of function of key genes involved in muscle function. Although there is until now no curative treatment to stop the progression of most myopathies, a significant number of experimental gene- and cell-based strategies and approaches have been and are being tested in vitro and in animal models, aiming to restore gene function. Genome editing using programmable endonucleases is a powerful tool for modifying target genome sequences and has been extensively used over the last decade to correct in vitro genetic defects of many single-gene diseases. By inducing double-strand breaks (DSBs), the engineered endonucleases specifically target chosen sequences. These DSBs are spontaneously repaired either by homologous recombination in the presence of a sequence template, or by nonhomologous-end joining error prone repair. In this review, we highlight recent developments and challenges for genome-editing based strategies that hold great promise for muscular dystrophies and regenerative medicine.

Reactive oxygen species are at the origin of muscular fatigue and atrophy. They are also linked to muscular dystrophies, a group of human genetic diseases. Several studies point to the benefits of application of antioxidants and uncouplers of oxidative phosphorylation to improve the functional activity of normal and pathological muscles. Other studies point to potential dangers of these compounds. Aim. To study the effect of mitochondria-targeted antioxidants and uncouplers of oxidative phosphorylation on muscle differentiation. Methods. Muscle differentiation was induced by serum starvation and monitored by troponin T staining. Results. the mitochondria-targeted uncoupler of oxidative phosphorylation C12TPP, but not the mitochondria-targeted antioxidant SkQ1, inhibit fusion of primary myoblasts upon their differentiation, but do not affect the synthesis of troponin T, a protein marker of muscle differentiation. Conclusion. The effect of C12TPP could be at least partially mediated by inhibition of reactive oxygen species (ROS) production since antioxidant N-acetylcysteine at high doses also inhibited differentiation of myoblasts. Активні форми кисню (АФК) можуть викликати м'язову втому і атрофію м'язів. АФК також пов'язані з м'язовими дистрофії. Безліч досліджень вказує на позитивний вплив антиоксидантів і разобщітелей окисного фосфорилювання на функціональну активність м'язів в нормі та патології. Мета. Вивчити вплив мітохондріальної-спрямованих антиоксидантів і разобщітелей окисного фосфорилювання на диференціювання первинних міобластів людини. Методи. Результати. мітохондріальної-спрямований разобщитель окисного фосфорилювання C12TPP, але не мітохондріальної-спрямований антиоксидант SkQ1, пригнічує злиття міобластів при диференціюванні, при цьому не впливаючи на експресію тропонина Т, білкового маркера м'язової диференціювання. Висновки. Вплив C12TPP може бути частково викликано пригніченням АФК, так як високі концентрації класичного антиоксиданту N-ацетилцистеїну також інгібували диференціювання міобластів людини. Активные формы кислорода (АФК) могут вызывать мышечную усталость и атрофию мышц. АФК также связаны с мышечными дистрофиями. Множество исследований указывает на положительное влияние антиоксидантов и разобщителей окислительного фосфорилирования на функциональную активность мышц в норме и патологии. Цель. Изучить влияние митохондриально-направленных антиоксидантов и разобщителей окислительного фосфорилирования на дифференцировку первичных миобластов человека. Методы. Результаты. митохондриально-направленный разобщитель окислительного фосфорилирования C12TPP, но не митохондриально-направленный антиоксидант SkQ1, ингибирует слияние миобластов при дифференцировке, при этом не влияя на экспрессию тропонина Т, белкового маркера мышечной дифференцировки. Выводы. Влияние C12TPP может быть частично вызвано ингибированием АФК, так как высокие концентрации классического антиоксиданта N-ацетилцистеина также ингибировали дифференцировку миобластов человека.

Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context. J. Cell. Physiol. 230: 62–71, 2016. © 2015 Wiley Periodicals, Inc.

International audience; Skeletal muscle is a highly oxygen-consuming tissue that ensures body support and movement, as well as nutrient and temperature regulation. DNA damage induced by reactive oxygen species is present in muscles and tends to accumulate with age. Here, we present a summary of data obtained on DNA damage and its implication in muscle homeostasis, myogenic differentiation and neuromuscular disorders. Controlled and transient DNA damage appears to be essential for muscular homeostasis and differentiation while uncontrolled and chronic DNA damage negatively affects muscle health.

Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy linked to a deletion of a subset of D4Z4 macrosatellite repeats accompanied by a chromatin relaxation of the D4Z4 array on chromosome 4q. In vitro, FSHD primary myoblasts show altered expression of oxidative-related genes and are more susceptible to oxidative stress. Double homeobox 4 (DUX4) gene, encoded within each D4Z4 unit, is normally transcriptionally silenced but is found aberrantly expressed in skeletal muscles of FSHD patients. Its expression leads to a deregulation of DUX4 target genes including those implicated in redox balance. Here, we assessed DNA repair efficiency of oxidative DNA damage in FSHD myoblasts and DUX4-transfected myoblasts. We have shown that the DNA repair activity is altered neither in FSHD myoblasts nor in immortalized human myoblasts transiently expressing DUX4. DNA damage caused by moderate doses of an oxidant is efficiently repaired while FSHD myoblasts exposed for 24 h to high levels of oxidative stress accumulated more DNA damage than normal myoblasts, suggesting that FSHD myoblasts remain more vulnerable to oxidative stress at high doses of oxidants.

International audience; Facioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Western world, however, its etiology remains only partially understood. Here, we provide evidence of constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage accumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition reduced the level of DNA damage in FSHD myoblasts. Addition of tempol, a powerful antioxidant, to the culture medium of proliferating DUX4-transfected myoblasts and FSHD myoblasts reduced the level of DNA damage, suggesting that DNA alterations are mainly due to oxidative stress. Antioxidant treatment during the myogenic differentiation of FSHD myoblasts significantly reduced morphological defects in myotube formation. We propose that the induction of DNA damage is a novel function of the DUX4 protein affecting myogenic differentiation of FSHD myoblasts.

Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context.

Human immunodeficiency virus-1 (HIV-1) Tat protein is one of the most important regulatory proteins for viral gene expression in the host cell and can modulate different cellular processes. In addition, Tat is secreted by the infected cell and can be internalized by neighboring cells; therefore, it affects both infected and uninfected cells. Tat can modulate cellular processes by interacting with different cellular structures and signaling pathways. In the nucleus, Tat might be localized either in the nucleoplasm or the nucleolus depending on its concentration. Here we review the distinct functions of Tat in the nucleoplasm and the nucleolus in connection with viral infection and HIV-induced oncogenesis.