Visualizing Muscle Sialic Acid Expression in the GNED207VTgGne-/- Cmah-/- Model of GNE Myopathy: A Comparison of Dietary and Gene Therapy Approaches.

BACKGROUND: GNE myopathy (GNEM) is a rare, adult-onset, inclusion body myopathy that results from mutations in the GNE gene. GNE encodes UDP-GlcNAc epimerase/ManNAc-6 kinase, a protein with two enzymatic activities that comprise the committed step in biosynthesis of sialic acid (SA), an essential glycan that appears on the terminal positions of many extracellular oligosaccharide chains. These GNE mutations can cause a reduction of SA in many tissues, although pathology is restricted to skeletal muscles through a poorly understood mechanism. OBJECTIVE: Despite recent advances in the field, it remains unclear which therapeutic avenue is most promising for the restoration of SA level in skeletal muscle affected by GNEM. Our objective was to assess dietary and gene therapy strategies for GNEM in Cmah-deficient GNED207VTgGne-/- mice, a model that allows for the visualization of orally delivered N-glycolylneuraminic acid (Neu5Gc), one of the two predominant SA forms in muscle. METHODS: Methods included in situ physiology studies of the tibialis anterior muscle, studies of ambulation and limb grip strength, and muscle staining using MAA, SNA, and anti-Neu5Gc antibody, along with qPCR, qRT-PCR, western blot, and HPLC studies to assess virally introduced DNA, GNE gene expression, GNE protein expression, and SA expression. RESULTS: We found that a diet enriched in Neu5Gc-containing glycoproteins had no impact on Neu5Gc immunostaining in muscles of GNEM model mice. Delivery of a single high dose oral Neu5Gc therapy, however, did increase Neu5Gc immunostaining, though to levels below those found in wild type mice. Delivery of a single dose of GNE gene therapy using a recombinant Adeno Associated Virus (rAAV) vector with a liver-specific or a muscle-specific promoter both caused increased muscle Neu5Gc immunostaining that exceeded that seen with single dose monosaccharide therapy. CONCLUSIONS: Our findings indicate that dietary loading of Neu5Gc-containing glycoproteins is not effective in increasing muscle Neu5Gc expression, while single dose oral Neu5Gc monosaccharide or GNE gene therapy are. Neu5Gc immunostaining, however, showed greater changes than did lectin staining or HPLC analysis. Taken together, these results suggest that Neu5Gc immunostaining may be more sensitive technique to follow SA expression than other more commonly used methods and that liver expression of GNE may contribute overall muscle SA content.

Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.

Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.

Sporadic Inclusion Body Myositis and Other Rimmed Vacuolar Myopathies.

PURPOSE OF REVIEW: This article reviews the clinical, laboratory, and histopathologic features of sporadic inclusion body myositis (IBM) and explores its pathogenic overlap with inherited myopathies that have IBM-like pathology. RECENT FINDINGS: Sporadic IBM is the most common acquired muscle disease in patients older than 50 years of age and is becoming more prevalent because of the increasing age of the population, the emerging development of more inclusive diagnostic criteria, and the advent of a diagnostic autoantibody. No effective therapy is known, and the pathogenic mechanism remains unclear. Some pathogenic insight can be gleaned from other myopathies with pathologic similarities or hereditary inclusion body myopathies. Although clinically distinct from sporadic IBM, preclinical models of hereditary inclusion body myopathy have offered an opportunity to move some therapies toward clinical development. SUMMARY: Patients with sporadic IBM experience significant morbidity, and the disease is associated with a large unmet medical need. As therapies are developed, improved diagnosis will be essential. Early diagnosis relies on awareness, clinical history, physical examination, laboratory features, and appropriate muscle biopsy processing. Future research is needed to understand the natural history, identify genetic risk factors, and validate biomarkers to track disease progression. These steps are essential as we move toward therapeutic interventions.

GNE myopathy: current update and future therapy.

GNE myopathy is an autosomal recessive muscle disease caused by biallelic mutations in GNE, a gene encoding for a single protein with key enzymatic activities, UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase, in sialic acid biosynthetic pathway. The diagnosis should be considered primarily in patients presenting with distal weakness (foot drop) in early adulthood (other onset symptoms are possible too). The disease slowly progresses to involve other lower and upper extremities' muscles, with marked sparing of the quadriceps. Characteristic findings on biopsies of affected muscles include 'rimmed' (autophagic) vacuoles, aggregation of various proteins and fibre size variation. The diagnosis is confirmed by sequencing of the GNE gene. Note that we use a new mutation nomenclature based on the longest transcript (GenBank: NM_001128227), which encodes a 31-amino acid longer protein than the originally described one (GenBank: NM_005476), which has been used previously in most papers. Based upon the pathophysiology of the disease, recent clinical trials as well as early gene therapy trials have evaluated the use of sialic acid or N-acetylmannosamine (a precursor of sialic acid) in patients with GNE myopathy. Now that therapies are under investigation, it is critical that a timely and accurate diagnosis is made in patients with GNE myopathy.

Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion.

Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in a delay and reduction in injury-triggered lysosomal exocytosis. We find repair of injured cells does not involve formation of intracellular membrane patch through lysosome-lysosome fusion; instead, individual lysosomes fuse with the injured cell membrane, releasing acid sphingomyelinase (ASM). ASM secretion was reduced in injured dysferlinopathic cells, and acute treatment with sphingomyelinase restored the repair ability of dysferlinopathic myoblasts and myofibers. Our results provide the mechanism for dysferlin-mediated repair of skeletal muscle sarcolemma and identify ASM as a potential therapy for dysferlinopathy.

Correction of the Middle Eastern M712T mutation causing GNE myopathy by trans-splicing.

GNE myopathy is a rare neuromuscular autosomal recessive disease, resulting from mutations in the gene UDP N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). The most frequent mutation is the single homozygous missense mutation, M712T-the Middle Eastern mutation-located ten amino acids before the end of the protein. We have used an adeno-associated virus (AAV)-based trans-splicing (TS) vector as a gene therapy tool to overcome this mutation by replacing the mutated last exon of GNE by the wild-type exon while preserving the natural endogenous regulatory machinery. We have designed relevant plasmids directed either to mouse or to human GNE. Following transfection of C2C12 murine muscle cells with the mouse TS vectors, we have been able to detect by nested RT-PCR trans-spliced molecules carrying the wild-type exon 12 of GNE. Similarly, transfection of HEK293 human cells with the human-directed TS vectors resulted in the generation of trans-spliced human GNE RNA molecules. Furthermore, infection of primary muscle cells from a GNE myopathy patient carrying the homozygous M712T mutation, with an AAV8-based viral vector carrying a human-directed TS construct, resulted in the generation of wild-type GNE transcripts in addition to the mutated ones. These studies provide a proof of concept that the TS approach could be used to partially correct the Middle Eastern mutation in GNE myopathy patients. These results provide the basis for in vivo research in animal models using the AAV platform with TS plasmids as a potential genetic therapy for GNE myopathy.

GNE myopathy: a personal trip from bedside observation to therapeutic trials.

The trip is not over yet as definite therapy for GNE myopathy is not yet available. Also the exact mechanisms by which GNE defects lead to isolated muscle disease in humans are not fully recognized. But in the Gaetano Conte lecture of 2013 I have tried to describe how much a progress was made in several research laboratories and clinical institutes in the investigation of this unique myopathy.

Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells.

The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides (AONs), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON-treated blood-derived CD133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/blAJ dysferlin null mice. We thus extended these experiments producing the full-length dysferlin mediated by a lentiviral vector in blood-derived CD133+ stem cells isolated from the same patients. Transplantation of engineered blood-derived CD133+ stem cells into scid/blAJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus-mediated delivery of full-length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies.

[Miyoshi distal muscular dystrophy (Miyoshi myopathy)].

We present an overview of autosomal recessive distal muscular dystrophy (ARDMD), including recent molecular genetic findings. ARDMD is often referred to as Miyoshi-type distal muscular dystrophy (MDMD) or Miyoshi myopathy (MM). The onset of MDMD occurs in early adulthood. Muscle atrophy is most dominant in distal leg muscles, especially the flexor muscles, i.e., gastrocnemius and soleus. As MDMD advances, muscle atrophy progresses to the thigh and hip muscles. Toe standing is impaired but heel standing can still be accomplished early in the disease course. This is followed by difficulty in standing and walking. The patients rarely become confined to bed. Serum creatine kinase level is markedly elevated, e.g., 100 times the upper limit of the normal range early in the disease course. Pre-symptomatic patients may also have high creatine kinase levels. Heterozygous individuals may have only slightly elevated creatine kinase levels. Recent development revealed that MDMD and LGMD2B are both caused by mutations in the dysferlin gene (DYSF). C1939G, G3370T, 3746delG, and 4870delT are reported to be common mutations among patients with MDMD. The dysferlin protein is presumably involved in the repair of muscle cell membranes. Among the patients reported originally by Miyoshi et al., 3 affected individuals from 3 different families were confirmed carriers of dysferlin mutations. Additionally, 1 heterozygous individual was identified. Although MDMD and LGMD2B are caused by the mutation of the same gene, ARDMD is characterized by initial involvement of leg flexors while LGMD2B is characterized by involvement of the proximal leg muscles. The difference in the distribution becomes obscure as the 2 diseases progress. The temporal profiles of functional impairment in the 2 diseases are reportedly very similar. When MDMD is suspected, it is important to carefully observe the relevant leg, more specially the flexor muscle group.

[Development of therapy for distal myopathy with rimmed vacuoles].

Distal myopathy with rimmed vacuoles (DMRV), also called hereditary inclusion body myopathy, is an autosomal recessive disorder caused by homozygous or compound heterozygous missense mutations in GNE which encodes a protein with two enzymatic activities in sialic acid biosynthesis: UDP-GlcNAc 2-epimerase and ManNAc kinase. The disease starts from 1540 years and is slowly progressive. DMRV preferentially affects tibialis anterior and hamstrings muscles, and has characteristic findings in muscle pathology which include rimmed vacuoles, tubulofilamentous inclusions, deposition of amyloid, and phosphorylated tau. We generated DMRV mice (Gne -/- hGNE D176V-Tg) by crossmating Gne knock-out heterozygous mouse and human GNE p.D176V transgenic mouse. This model mouse recapitulates DMRV clinically, pathologically, and biochemically by developing muscle weakness and atrophy from 21 weeks, amyloid deposition from 31 weeks, and rimmed vacuoles and phosphorylated tau from 41 weeks while having lifelong hyposialylation. We gave three types of GNE metabolites, ManNAc, NeuAc and sialyllactose, to DMRV mice orally from 15 weeks until 55 weeks of age. Sialic acid supplementation almost completely precluded the disease and virtually no sign of DMRV was seen even at 55 weeks of age, indicating that decreased sialic acid is the cause of myopathic phenotype and sialic acid supplementation can prevent the disease process.

Hereditary inclusion-body myopathy: clues on pathogenesis and possible therapy.

Hereditary inclusion-body myopathy (h-IBM), or distal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with onset in early adult life and a progressive course leading to severe disability. h-IBM/DMRV is due to mutations of a gene (GNE) that codes for a rate-limiting enzyme in the sialic acid biosynthetic pathway. Despite the identification of the causative gene defect, it has not been unambiguously clarified how GNE gene mutations impair muscle metabolism. Although numerous studies have indicated a key role of hyposialylation of glycoproteins in h-IBM/DMRV pathogenesis, others have demonstrated new and unpredicted functions of the GNE gene, outside the sialic acid biosynthetic pathway, that may also be relevant. This review illustrates the clinical and pathologic characteristics of h-IBM/DMRV and the main clues available to date concerning the possible pathogenic mechanisms and therapeutic perspectives of this disorder.

Recent advances in distal myopathy with rimmed vacuoles (DMRV) or hIBM: treatment perspectives.

PURPOSE OF REVIEW: Distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy is an adult-onset autosomal recessive, slowly progressive and debilitating myopathy due to mutations in the gene that regulates the synthesis of sialic acid. This review aims to update our knowledge of this myopathy and to review studies about pathomechanism and therapeutic strategies. RECENT FINDINGS: Owing to the mutated gene, it was expected that the pathomechanism of this myopathy would be based on hyposialylation, a highly controversial phenomenon. This concept has been supported by findings in two recently generated animal models. In addition, the intracellular amyloid-beta accumulation in a distal myopathy with rimmed vacuole mouse model is relevant to similar findings in patients. SUMMARY: Clarifying the role of hyposialylation in distal myopathy with rimmed vacuole/hereditary inclusion body myopathy could potentially lead to a therapeutic strategy for this progressive myopathy. In addition, strategies aimed at preventing amyloid-beta deposition or enhancing its clearance could also be beneficial, as this epiphenomenon is now known to occur early in the course of the disease.

Collagen VI related muscle disorders.

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two conditions which were previously believed to be completely separate entities. BM is a relatively mild dominantly inherited disorder characterised by proximal weakness and distal joint contractures. UCMD was originally described as an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. Here we review the clinical phenotypes of BM and UCMD and their diagnosis and management, and provide an overview of the current knowledge of the pathogenesis of collagen VI related disorders.