Generation of a lymphoblastoid-derived induced pluripotent stem cell line (CBRCULi015-A) from a patient with congenital myotonic dystrophy.

Congenital myotonic dystrophy (CDM) is a genetic disease caused by an abnormally long CTG repeat expansion in the DMPK gene, which generally increases in size following intergenerational transmission. CDM is the rarest and most severe form of myotonic dystrophy type 1, yet an important number of patient-derived cells are needed to study this heterogeneous disease. Therefore, we have reprogrammed lymphoblastoid cells derived from a 3-year-old male with CDM into induced pluripotent stem cells (iPSCs; CBRCULi015-A) featuring 1800 CTG repeats and characterized their pluripotent state. This cell line constitutes an important resource to study CDM and potential treatments in vitro.

Generation of induced pluripotent stem cell lines from pediatric patients with congenital myotonic dystrophy (CBRCULi012-A and CBRCULi013-A) and age-matched controls (CBRCULi010-A and CBRCULi011-A)

Congenital myotonic dystrophy (CDM) is an autosomal dominant multisystemic disorder attributed to a large expansion of CTG trinucleotide repeats within the myotonic dystrophy protein kinase (DMPK) gene. In this study, we successfully reprogrammed dermal fibroblasts derived from two pediatric CDM patients and two age-matched individuals into induced pluripotent stem cells (iPSCs) using a non-integrating viral vector. The resulting CDM iPSC lines harbored approximately 2000 CTG repeats in the mutated DMPK allele. These iPSC lines expressed pluripotency markers and exhibited the capacity to differentiate into cells representing all three germinal layers, confirming their reliability as a research tool for investigating CDM and therapeutic strategies.

In vitro glycation of a tissue-engineered wound healing model to mimic diabetic ulcers.

Diabetic foot ulcers are a major complication of diabetes that occurs following minor trauma. Diabetes-induced hyperglycemia is a leading factor inducing ulcer formation and manifests notably through the accumulation of advanced glycation end-products (AGEs) such as N-carboxymethyl-lysin. AGEs have a negative impact on angiogenesis, innervation, and reepithelialization causing minor wounds to evolve into chronic ulcers which increases the risks of lower limb amputation. However, the impact of AGEs on wound healing is difficult to model (both in vitro on cells, and in vivo in animals) because it involves a long-term toxic effect. We have developed a tissue-engineered wound healing model made of human keratinocytes, fibroblasts, and endothelial cells cultured in a collagen sponge biomaterial. To mimic the deleterious effects induced by glycation on skin wound healing, the model was treated with 300 µM of glyoxal for 15 days to promote AGEs formation. Glyoxal treatment induced carboxymethyl-lysin accumulation and delayed wound closure in the skin mimicking diabetic ulcers. Moreover, this effect was reversed by the addition of aminoguanidine, an inhibitor of AGEs formation. This in vitro diabetic wound healing model could be a great tool for the screening of new molecules to improve the treatment of diabetic ulcers by preventing glycation.

Recent Progress and Challenges in the Development of Antisense Therapies for Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a dominant genetic disease in which the expansion of long CTG trinucleotides in the 3' UTR of the myotonic dystrophy protein kinase (DMPK) gene results in toxic RNA gain-of-function and gene mis-splicing affecting mainly the muscles, the heart, and the brain. The CUG-expanded transcripts are a suitable target for the development of antisense oligonucleotide (ASO) therapies. Various chemical modifications of the sugar-phosphate backbone have been reported to significantly enhance the affinity of ASOs for RNA and their resistance to nucleases, making it possible to reverse DM1-like symptoms following systemic administration in different transgenic mouse models. However, specific tissue delivery remains to be improved to achieve significant clinical outcomes in humans. Several strategies, including ASO conjugation to cell-penetrating peptides, fatty acids, or monoclonal antibodies, have recently been shown to improve potency in muscle and cardiac tissues in mice. Moreover, intrathecal administration of ASOs may be an advantageous complementary administration route to bypass the blood-brain barrier and correct defects of the central nervous system in DM1. This review describes the evolution of the chemical design of antisense oligonucleotides targeting CUG-expanded mRNAs and how recent advances in the field may be game-changing by forwarding laboratory findings into clinical research and treatments for DM1 and other microsatellite diseases.

Enhanced Delivery of Ligand-Conjugated Antisense Oligonucleotides (C16-HA-ASO) Targeting Dystrophia Myotonica Protein Kinase Transcripts for the Treatment of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder that affects many organs. It is caused by the expansion of a cytosine-thymine-guanine triplet repeat in the 3' untranslated region of the human dystrophia myotonica protein kinase (hDMPK) gene, which results in a toxic gain of function of mutant hDMPK RNA transcripts. Antisense oligonucleotides (ASOs) have emerged in recent years as a potential gene therapy to treat DM1. However, the clinical efficacy of the systemic administration of ASOs is limited by a combination of insufficient potency and poor tissue distribution. In the present study, we assessed the potential of a new ligand-conjugated ASO (IONIS-877864; C16-HA-ASO) to target mutant hDMPK mRNA transcripts in the DMSXL mouse model of DM1 carrying over 1000 CTG pathogenic repeats. DMSXL mice were treated subcutaneously for 9 weeks with either IONIS-877864 (12.5 or 25 mg/kg) or IONIS-486178 (12.5 or 25 mg/kg), an unconjugated ASO with the same sequence. At 25 mg/kg, IONIS-877864 significantly enhanced ASO delivery into the striated muscles of DMSXL mice following systemic administration compared with the unconjugated control. IONIS-877864 was also more efficacious than IONIS-486178, reducing mutant hDMPK transcripts by up to 92% in the skeletal muscles and 78% in the hearts of DMSXL mice. The decrease in mutant hDMPK transcripts in skeletal muscles caused by IONIS-877864 was associated with a significant improvement in muscle strength. IONIS-877864 was nontoxic in the DMSXL mouse model. The present study showed that the C16-HA-conjugated ASO is a powerful tool for the development of gene therapy for DM1.

Antisense oligonucleotides as a potential treatment for brain deficits observed in myotonic dystrophy type 1.

Myotonic dystrophy, or dystrophia myotonica type 1 (DM1), is a multi-systemic disorder and is the most common adult form of muscular dystrophy. It affects not only muscles but also many organs, including the brain. Cerebral impairments include cognitive deficits, daytime sleepiness, and loss of visuospatial and memory functions. The expression of mutated transcripts with CUG repeats results in a gain of toxic mRNA function. The antisense oligonucleotide (ASO) strategy to treat DM1 brain deficits is limited by the fact that ASOs do not cross the blood-brain barrier after systemic administration, indicating that other methods of delivery should be considered. ASO technology has emerged as a powerful tool for developing potential new therapies for a wide variety of human diseases, and its potential has been proven in a recent clinical trial. Targeting DMPK mRNA in neural cells derived from human induced pluripotent stem cells obtained from a DM1 patient with the IONIS 486178 ASO abolished CUG-expanded foci, enabled nuclear redistribution of MBNL1/2, and corrected aberrant splicing. Intracerebroventricular injection of the IONIS 486178 ASO in DMSXL mice decreased the levels of mutant DMPK mRNAs by up to 70% throughout different brain regions. It also reversed behavioral abnormalities following neonatal administration. The present study indicated that the IONIS 486178 ASO targets mutant DMPK mRNAs in the brain and strongly supports the feasibility of a therapy for DM1 patients based on the intrathecal injection of an ASO.

Deciphering the mechanisms underlying brain alterations and cognitive impairment in congenital myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is a multisystemic and heterogeneous disorder caused by the expansion of CTG repeats in the 3' UTR of the myotonic dystrophy protein kinase (DMPK) gene. There is a congenital form (CDM1) of the disease characterized by severe hypotonia, respiratory insufficiency as well as developmental delays and intellectual disabilities. CDM1 infants manifest important brain structure abnormalities present from birth while, in contrast, older patients with adult-onset DM1 often present neurodegenerative features and milder progressive cognitive deficits. Promising therapies targeting central molecular mechanisms contributing to the symptoms of adult-onset DM1 are currently in development, but their relevance for treating cognitive impairment in CDM1, which seems to be a partially distinct neurodevelopmental disorder, remain to be elucidated. Here, we provide an update on the clinical presentation of CDM1 and review recent in vitro and in vivo models that have provided meaningful insights on its consequences in development, with a particular focus on the brain. We discuss how enhanced toxic gain-of-function of the mutated DMPK transcripts with larger CUG repeats and the resulting dysregulation of RNA-binding proteins may affect the developing cortex in utero. Because the methylation of CpG islets flanking the trinucleotide repeats has emerged as a strong biomarker of CDM1, we highlight the need to investigate the tissue-specific impacts of these chromatin modifications in the brain. Finally, we outline promising potential therapeutic treatments for CDM1 and propose future in vitro and in vivo models with great potential to shed light on this disease.

Development of an innervated tissue-engineered skin with human sensory neurons and Schwann cells differentiated from iPS cells.

Cutaneous innervation is increasingly recognized as a major element of skin physiopathology through the neurogenic inflammation driven by neuropeptides that are sensed by endothelial cells and the immune system. To investigate this process in vitro, models of innervated tissue-engineered skin (TES) were developed, yet exclusively with murine sensory neurons extracted from dorsal root ganglions. In order to build a fully human model of innervated TES, we used induced pluripotent stem cells (iPSC) generated from human skin fibroblasts. Nearly 100% of the iPSC differentiated into sensory neurons were shown to express the neuronal markers BRN3A and ß3-tubulin after 19 days of maturation. In addition, these cells were also positive to TRPV1 and neurofilament M, and some of them expressed Substance P, TrkA and TRPA1. When stimulated with molecules inducing neuropeptide release, iPSC-derived neurons released Substance P and CGRP, both in conventional monolayer culture and after seeding in a 3D fibroblast-populated collagen sponge model. Schwann cells, the essential partners of neurons for function and axonal migration, were also successfully differentiated from human iPSC as shown by their expression of the markers S100, GFAP, p75 and SOX10. When cultured for one additional month in the TES model, iPSC-derived neurons seeded at the bottom of the sponge formed a network of neurites spanning the whole TES up to the epidermis, but only when combined with mouse or iPSC-derived Schwann cells. This unique model of human innervated TES should be highly useful for the study of cutaneous neuroinflammation. STATEMENT OF SIGNIFICANCE: The purpose of this work was to develop in vitro an innovative fully human tissue-engineered skin enabling the investigation of the influence of cutaneous innervation on skin pathophysiology. To reach that aim, neurons were differentiated from human induced pluripotent stem cells (iPSCs) generated from normal human skin fibroblasts. This innervated tissue-engineered skin model will be the first one to show iPSC-derived neurons can be successfully used to build a 3D nerve network in vitro. Since innervation has been recently recognized to play a central role in many human skin diseases, such as psoriasis and atopic dermatitis, this construct promises to be at the forefront to model these diseases while using patient-derived cells.