Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (µDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-µDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; µDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

Micro-dystrophin AAV Vectors Made by Transient Transfection and Herpesvirus System Are Equally Potent in Treating mdx Mouse Muscle Disease.

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method.

Membrane recruitment of nNOSµ in microdystrophin gene transfer to enhance durability.

Several gene transfer clinical trials are currently ongoing with the common aim of delivering a shortened version of dystrophin, termed a microdystrophin, for the treatment of Duchenne muscular dystrophy (DMD). However, one of the main differences between these trials is the microdystrophin protein produced following treatment. Each gene transfer product is based on different selections of dystrophin domain combinations to assemble microdystrophin transgenes that maintain functional dystrophin domains and fit within the packaging limits of an adeno-associated virus (AAV) vector. While domains involved in mechanical function, such as the actin-binding domain and ß-dystroglycan binding domain, have been identified for many years and included in microdystrophin constructs, more recently the neuronal nitric oxide synthase (nNOS) domain has also been identified due to its role in enhancing nNOS membrane localization. As nNOS membrane localization has been established as an important requirement for prevention of functional ischemia in skeletal muscle, inclusion of the nNOS domain into a microdystrophin construct represents an important consideration. The aim of this mini review is to highlight what is currently known about the nNOS domain of dystrophin and to describe potential implications of this domain in a microdystrophin gene transfer clinical trial.

A Five-Repeat Micro-Dystrophin Gene Ameliorated Dystrophic Phenotype in the Severe DBA/2J-mdx Model of Duchenne Muscular Dystrophy.

Micro-dystrophins are highly promising candidates for treating Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. Here, we report robust disease rescue in the severe DBA/2J-mdx model with a neuronal nitric oxide synthase (nNOS)-binding micro-dystrophin vector. 2 × 1013 vector genome particles/mouse of the vector were delivered intravenously to 10-week-old mice and were evaluated at 6 months of age. Saturated micro-dystrophin expression was detected in all skeletal muscles and the heart and restored the dystrophin-associated glycoprotein complex and nNOS. In skeletal muscle, therapy substantially reduced fibrosis and calcification and significantly attenuated inflammation. Centronucleation was significantly decreased in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles but not in the quadriceps. Muscle function was normalized in the TA and significantly improved in the EDL muscle. Heart histology and function were also evaluated. Consistent with the literature, DBA/2J-mdx mice showed myocardial calcification and fibrosis and cardiac hemodynamics was compromised. Surprisingly, similar myocardial pathology and hemodynamic defects were detected in control DBA/2J mice. As a result, interpretation of the cardiac data proved difficult due to the confounding phenotype in control DBA/2J mice. Our results support further development of this microgene vector for clinical translation. Further, DBA/2J-mdx mice are not good models for Duchenne cardiomyopathy.

Small Fractions of Muscular Dystrophy Embryonic Stem Cells Yield Severe Cardiac and Skeletal Muscle Defects in Adult Mouse Chimeras.

Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.

Neuronal nitric oxide synthase localizes to utrophin expressing intercalated discs and stabilizes their structural integrity.

The neuronal nitric-oxide synthase (nNOS) splice variant nNOSµ is essential for skeletal muscle function. Its localization is dependent on dystrophin, which stabilizes the dystrophin glycoprotein complex (DGC) at the sarcolemma of skeletal muscle fibers. In Duchenne muscular dystrophy (DMD) dystrophin is absent and sarcolemmal nNOS is lost. This leads to functional ischemia due to a decrease in contraction-induced vasodilation. In cardiomyocytes, nNOSµ is believed to be the predominant NOS isoform. However, the association of nNOS with the DGC in the heart is unclear. Here, we report nNOS localization at the intercalated discs (IDs) of cardiomyocytes, where utrophin is highly expressed. In mdx, mdx:utr, nNOSµ knock-out (KO), and mdx:nNOSµ KO mice, we observed a gradual reduction of nNOS at IDs and disrupted ID morphology, compared to wild-type. In mdx:nNOSµ KO mice, but not in mdx or nNOSµ KO mice, we also observed an early development of cardiac fibrosis. These findings suggest that nNOS localization in the heart may not depend exclusively on the presence of dystrophin. Additionally, the ß1 subunit of soluble guanylyl cyclase (sGC), responsible for the production of cGMP through nitric oxide (NO) signaling, was also detected at the IDs. Together, our results suggest a new role of nNOS at the IDs for the cGMP-dependent NO pathway and the maintenance of ID morphology.

Reversible mitochondrial DNA accumulation in nuclei of pluripotent stem cells.

According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

Increased sarcolipin expression and decreased sarco(endo)plasmic reticulum Ca2+ uptake in skeletal muscles of mouse models of Duchenne muscular dystrophy.

Abnormal intracellular Ca(2+) handling is an important factor in the progressive functional decline of dystrophic muscle. In the present study, we investigated the function of sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA) in various dystrophic muscles of mouse models of Duchenne muscular dystrophy. Our studies show that the protein expression of sarcolipin, a key regulator of the SERCA pump is abnormally high and correlates with decreased maximum velocity of SR Ca(2+) uptake in the soleus, diaphragm and quadriceps of mild (mdx) and severe (mdx:utr-/-) dystrophic mice. These changes are more pronounced in the muscles of mdx:utr-/- mice. We also found increased expression of SERCA2a and calsequestrin specifically in the dystrophic quadriceps. Immunostaining analysis further showed that SERCA2a expression is associated both with fibers expressing slow-type myosin and regenerating fibers expressing embryonic myosin. Together, our data suggest that sarcolipin upregulation is a common secondary alteration in all dystrophic muscles and contributes to the abnormal elevation of intracellular Ca(2+) concentration via SERCA inhibition.

Nitric oxide signalling pathway in Duchenne muscular dystrophy mice: up-regulation of L-arginine transporters.

DMD (Duchenne muscular dystrophy) is an incurable rapidly worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle a lack of dystrophin disrupts the recruitment of neuronal NOS (nitric oxide synthase) to the sarcolemma thus affecting NO (nitric oxide) production. Utrophin is a dystrophin homologue, the expression of which is greatly up-regulated in the sarcolemma of dystrophin-negative fibres from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signalling pathway in the cardiac muscle of DMD patients. Thus we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin-/- (double knockout) mice (mdx:utr) to study key steps in NO signalling: L-arginine transporters, NOS and sGC (soluble guanylyl cyclase). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this nNOS activity was markedly decreased in both mdx and mdx:utr mice, whereas nNOS expression was only decreased in mdx:utr mouse hearts, suggesting that utrophin up-regulation in cardiomyocytes maintains nNOS levels, but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

IGF-II promotes stemness of neural restricted precursors.

Insulin-like growth factor (IGF)-I and IGF-II regulate brain development and growth through the IGF type 1 receptor (IGF-1R). Less appreciated is that IGF-II, but not IGF-I, activates a splice variant of the insulin receptor (IR) known as IR-A. We hypothesized that IGF-II exerts distinct effects from IGF-I on neural stem/progenitor cells (NSPs) via its interaction with IR-A. Immunofluorescence revealed high IGF-II in the medial region of the subventricular zone (SVZ) comprising the neural stem cell niche, with IGF-II mRNA predominant in the adjacent choroid plexus. The IGF-1R and the IR isoforms were differentially expressed with IR-A predominant in the medial SVZ, whereas the IGF-1R was more abundant laterally. Similarly, IR-A was more highly expressed by NSPs, whereas the IGF-1R was more highly expressed by lineage restricted cells. In vitro, IGF-II was more potent in promoting NSP expansion than either IGF-I or standard growth medium. Limiting dilution and differentiation assays revealed that IGF-II was superior to IGF-I in promoting stemness. In vivo, NSPs propagated in IGF-II migrated to and took up residence in periventricular niches while IGF-I-treated NSPs predominantly colonized white matter. Knockdown of IR or IGF-1R using shRNAs supported the conclusion that the IGF-1R promotes progenitor proliferation, whereas the IR is important for self-renewal. Q-PCR revealed that IGF-II increased Oct4, Sox1, and FABP7 mRNA levels in NSPs. Our data support the conclusion that IGF-II promotes the self-renewal of neural stem/progenitors via the IR. By contrast, IGF-1R functions as a mitogenic receptor to increase precursor abundance.

Dystrophin-compromised sarcoglycan-δ-knockout diaphragm requires full wild-type embryonic stem cell reconstitution for correction.

Limb-girdle muscular dystrophy-2F (LGMD-2F) is an incurable degenerative muscle disorder caused by a mutation in the sarcoglycan-δ (SGδ)-encoding gene (SGCD in humans). The lack of SGδ results in the complete disruption of the sarcoglycan complex (SGC) in the skeletal and cardiac muscle within the larger dystrophin-glycoprotein complex (DGC). The long-term consequences of SG ablation on other members of the DGC are currently unknown. We produced mosaic mice through the injection of wild-type (WT) embryonic stem cells (ESCs) into SGδ-knockout (KO) blastocysts. ESC-derived SGδ was supplied to the sarcolemma of 18-month-old chimeric muscle, which resulted in the restoration of the SGC. Despite SGC rescue, and contrary to previous observations obtained with WT/mdx chimeras (a mouse rescue paradigm for Duchenne muscular dystrophy), low levels of ESC incorporation were insufficient to produce histological corrections in SGδ-KO skeletal muscle or heart. The inefficient process of ESC rescue was more evident in the SGδ-KO diaphragm, which had reduced levels of dystrophin and no compensatory utrophin, and needed almost full WT ESC reconstitution for histological improvement. The results suggest that the SGδ-KO mouse model of LGMD is not amenable to ESC treatment.

Differential requirement for utrophin in the induced pluripotent stem cell correction of muscle versus fat in muscular dystrophy mice.

Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.

Developmental ablation of Id1 and Id3 genes in the vasculature leads to postnatal cardiac phenotypes.

The Id1 and Id3 genes play major roles during cardiac development, despite their expression being confined to non-myocardial layers (endocardium-endothelium-epicardium). We previously described that Id1Id3 double knockout (dKO) mouse embryos die at mid-gestation from multiple cardiac defects, but early lethality precluded the studies of the roles of Id in the postnatal heart. To elucidate postnatal roles of Id genes, we ablated the Id3 gene and conditionally ablated the Id1 gene in the endothelium to generate conditional KO (cKO) embryos. We observed cardiac phenotypes at birth and at 6 months of age. Half of the Id cKO mice died at birth. Postnatal demise was associated with cardiac enlargement and defects in the ventricular septum, trabeculation and vasculature. Surviving Id cKO mice exhibited fibrotic vasculature, cardiac enlargement and decreased cardiac function. An abnormal vascular response was also observed in the healing of excisional skin wounds of Id cKO mice. Expression patterns of vascular, fibrotic and hypertrophic markers were altered in the Id cKO hearts, but addition of Insulin-Like Growth Factor binding protein-3 (IGFbp3) reversed gene expression profiles of vascular and fibrotic, but not hypertrophic markers. Thus, ablation of Id genes in the vasculature leads to distinct postnatal cardiac phenotypes. These findings provide important insights into the role/s of the endocardial network of the endothelial lineage in the development of cardiac disease, and highlight IGFbp3 as a potential link between Id and its vascular effectors.

Injection of wild type embryonic stem cells into Mst1 transgenic blastocysts prevents adult-onset cardiomyopathy.

Embryonic stem cells have the capacity to differentiate into a wide range of cell types. We previously described that blastocyst injection of wild type (WT) embryonic stem cells (ESCs) into various knockout (KO) mouse models of human disease prevents disease from occurring. In this study we ask if the blastocyst approach can also correct defects in a mouse model of transgenic (Tg) overexpression of a pro-apoptotic factor. We injected ROSA26 (LacZ-marked) WT ESCs into human mammalian sterile 20 like-kinase 1 (Mst1) Tg blastocysts. Mst1 Tg mice overexpress Mst1, a pro-apoptotic factor, in a cardiac-specific manner. As a result, Mst1 Tg mice develop adult dilated cardiomyopathy driven by apoptosis, reduction in cell density and no hypertrophic compensation. Incorporation of WT ESCs generated WT/Mst1 chimeric mice with normal hearts at histological and functional levels. Accordingly, apoptosis and cell density parameters were normalized. The experiments suggest that an adult-onset cardiac myopathy induced by overexpression of the pro-apoptotic Mst1 can be reversed by developmental incorporation of WT ESCs. The findings also suggest that since forced expression of the Mst1 transgene is not abolished in the rescued chimeras, the WT ES-derived cells normalize pathways that lie downstream of Mst1. The results expand the therapeutic capability of the ESCs to mouse models that overproduce detrimental proteins.

Rescue of developmental defects by blastocyst stem cell injection: towards elucidation of neomorphic corrective pathways.

Stem cell-based therapy is an exciting area of high potential for regenerative medicine. To study disease prevention, we inject mouse embryonic stem cells (ESCs) into a variety of mouse blastocysts, most of which harbor mutations. Mice derived from these mutant blastocysts develop human-like diseases, either at developmental stages or in the adult, but blastocyst injection of ESCs prevents disease from occurring. Rather than entirely repopulating the affected organs, with just 20% of chimerism, the ESCs replenish protein levels that are absent in mutant mice, and induce novel or "neomorphic" signals that help circumvent the requirements for the mutations. We also show data indicating that the "neomorphic" mechanisms arise as a result of blastocyst injection of ESCs, regardless of the nature of the host blastocyst (mutant or wild-type). Thus, blastocyst injection of ESCs not only allows the study of disease prevention, but also unveils novel pathways whose activation may aid in the correction of congenital or acquired disease.

Blastocyst injection of embryonic stem cells: a simple approach to unveil mechanisms of corrections in mouse models of human disease.

Embryonic stem cell (ESC) research is a promising area of investigation with enormous therapeutic potential. We have injected murine wild type (WT) ESCs into a variety of mutant murine blastocysts, which are predisposed to develop a human-like disease, such as muscular dystrophy or the embryonic lethal "thin myocardial syndrome". In this review, we summarize data indicating that partial incorporation of ESCs is sufficient to prevent disease from occurring. We also present data indicating that blastocyst incorporation of ESCs may aid in the prevention of heart failure in stressed WT mice. In some cases, the rescue observed is predominantly non-cell autonomous and relies on the production of secreted factors from the ES-derived cells, but in others, cell replacement is required. Thus, congenital or acquired disease can be pre-emptively averted in mice by developmental injection of ESCs.