Local overexpression of the myostatin propeptide increases glucose transporter expression and enhances skeletal muscle glucose disposal.

Insulin resistance (IR) in skeletal muscle is a prerequisite for type 2 diabetes and is often associated with obesity. IR also develops alongside muscle atrophy in older individuals in sarcopenic obesity. The molecular defects that underpin this syndrome are not well characterized, and there is no licensed treatment. Deletion of the transforming growth factor-ß family member myostatin, or sequestration of the active peptide by overexpression of the myostatin propeptide/latency-associated peptide (ProMyo) results in both muscle hypertrophy and reduced obesity and IR. We aimed to establish whether local myostatin inhibition would have a paracrine/autocrine effect to enhance glucose disposal beyond that simply generated by increased muscle mass, and the mechanisms involved. We directly injected adeno-associated virus expressing ProMyo in right tibialis cranialis/extensor digitorum longus muscles of rats and saline in left muscles and compared the effects after 17 days. Both test muscles were increased in size (by 7 and 11%) and showed increased radiolabeled 2-deoxyglucose uptake (26 and 47%) and glycogen storage (28 and 41%) per unit mass during an intraperitoneal glucose tolerance test. This was likely mediated through increased membrane protein levels of GLUT1 (19% higher) and GLUT4 (63% higher). Interestingly, phosphorylation of phosphoinositol 3-kinase signaling intermediates and AMP-activated kinase was slightly decreased, possibly because of reduced expression of insulin-like growth factor-I in these muscles. Thus, myostatin inhibition has direct effects to enhance glucose disposal in muscle beyond that expected of hypertrophy alone, and this approach may offer potential for the therapy of IR syndromes.

Utilisation of specialist epilepsy services and antiseizure medication adherence rates in a cohort of people with epilepsy (PWE) accessing emergency care.

BACKGROUND: An epilepsy-related attendance at A&E is associated an increased risk of subsequent death within 6 months. Although further work is required to provide a definitive explanation to account for these findings, in the interim it would seem reasonable that services are designed to ensure timely access and provide support at a time of greatest risk. We aim to determine the frequency of patients accessing specialist neurology services following an epilepsy-related admission/unscheduled care episode and consider ASM adherence at the point of attendance. METHODS: Patients were identified retrospectively via the NHS Greater Glasgow and Clyde live integrated epilepsy Dashboard following an unscheduled epilepsy-related admission or A&E attendance between 1st January 2022 and 30th June 2022. We calculated adherence to anti-seizure medication for a period of 6 months prior to admission and defined poor medication adherence as a medication possession ratio of less than 80 %. We evaluated the rate of any outpatient neurology clinic attendance in the subsequent 3, 6 and 12 months following an epilepsy-related unscheduled care episode. Additional clinical information was identified via the electronic patient records. RESULTS: Between 1st Jan 2022 and 30th June 2022, there were 266 emergency care seizure-related attendances. The mean age at attendance was 46 years (range: 16-91). Most of PWE were males (63 %) and 37 % were females. Epilepsy classification-29.3 % had GGE, 41.7% had focal epilepsy, and in 29 % of cases the epilepsy was unclassified. Of the admissions, 107/ 266 (40.2 %) generated follow-up within 6 months of attendance. Poor medication adherence was noted in 54/266 (20.3 %). 28.2 % of cases had input from on-call neurology service during admission/ED attendance, and of those 60 % had ASM adjusted. 18 % of attendances had a background diagnosis of learning disability. One-third of attendances of PWE had a history of mental health disorder 35 % (93/266). 25 % of ED attendances noted an active history of alcohol consumption misuse or/and recreational drug use. 14 (5.5 %) of PWE died during the period of interest (12 months following the last ED visit). In 6/14 (42.3 %) death was associated with poor medication adherence. CONCLUSION: This study demonstrates that a significant proportion of patients who experienced seizure-related admissions/ attendance did not access specialist neurology services in a timely manner. In addition, poor medication adherence remains a problem for a substantial number of people living with epilepsy. Early access to specialist services may go some way to improving care and reducing excessive mortality in PWE by allowing anti-seizure medication to be titrated and poor medication adherence to be addressed in those at greatest risk.

Intercellular adhesion mediated by human muscle neural cell adhesion molecule: effects of alternative exon use.

Mouse 3T3 fibroblasts were permanently transfected with cDNAs encoding isoforms of the neural cell adhesion molecule (N-CAM) present in human skeletal muscle and brain. Parental and transfected cells were then used in a range of adhesion assays. In the absence of external shear forces, transfection with cDNAs encoding either transmembrane or glycosylphosphatidylinositol (GPI)-linked N-CAM species significantly increased the intercellular adhesiveness of 3T3 cells in suspension. Transfection of a cDNA encoding a secreted N-CAM isoform was without effect on adhesion. Cells transfected with cDNAs containing or lacking the muscle-specific domain 1 sequence, a four-exon group spliced into the muscle but not the brain GPI-linked N-CAM species, were equally adhesive in the assays used. We also demonstrate that N-CAM-mediated intercellular adhesiveness is inhibited by 0.2 mg/ml heparin; but, at higher concentrations, reduced adhesion of parental cells was also seen. Coaggregation of fluorescently labeled and unlabeled cell populations was performed and measured by comparing their distribution within aggregates with distributions that assume nonspecific (random) aggregation. These studies demonstrate that random aggregation occurs between transfected cells expressing the transmembrane and GPI-linked N-CAM species and between parental cells and those expressing the secreted N-CAM isoform. Other combinations of these populations tested exhibited partial adhesive specificity, indicating homophilic binding between surface-bound N-CAM. Thus, the approach exploited here allows for a full analysis of the requirements, characteristics, and specificities of the adhesive behavior of individual N-CAM isoforms.

Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines.

mAbs have been raised against different epitopes on the protein product of the DMDL gene, which is an autosomal homologue of the X-linked DMD gene for dystrophin. These antibodies provide direct evidence that DMDL protein is localized near acetylcholine receptors at neuromuscular junctions in normal and mdx mouse intercostal muscle. The primary location in tissues other than skeletal muscle is smooth muscle, especially in the vascular system, which may account for the wide tissue distribution previously demonstrated by Western blotting. The DMDL protein was undetectable in the nonjunctional sarcolemma of normal human muscle, but was observed in nonjunctional sarcolemma of Duchenne muscular dystrophy patients, where dystrophin itself is absent or greatly reduced. The expression of DMDL protein is not restricted to smooth and skeletal muscle, however, since relatively large amounts are present in transformed brain cell lines of both glial and Schwann cell origin. This contrasts with the low levels of DMDL protein in adult brain tissue.

Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms.

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.

Next-generation muscle-directed gene therapy by in silico vector design.

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cis-regulatory modules (CRMs), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy.

Comparative analysis of antisense oligonucleotide sequences for targeted skipping of exon 51 during dystrophin pre-mRNA splicing in human muscle.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in the absence of functional protein. In the majority of cases these are out-of-frame deletions that disrupt the reading frame. Several attempts have been made to restore the dystrophin mRNA reading frame by modulation of pre-mRNA splicing with antisense oligonucleotides (AOs), demonstrating success in cultured cells, muscle explants, and animal models. We are preparing for a phase I/IIa clinical trial aimed at assessing the safety and effect of locally administered AOs designed to inhibit inclusion of exon 51 into the mature mRNA by the splicing machinery, a process known as exon skipping. Here, we describe a series of systematic experiments to validate the sequence and chemistry of the exon 51 AO reagent selected to go forward into the clinical trial planned in the United Kingdom. Eight specific AO sequences targeting exon 51 were tested in two different chemical forms and in three different preclinical models: cultured human muscle cells and explants (wild type and DMD), and local in vivo administration in transgenic mice harboring the entire human DMD locus. Data have been validated independently in the different model systems used, and the studies describe a rational collaborative path for the preclinical selection of AOs for evaluation in future clinical trials.

Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.

PABPN1 gene therapy for oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.

Strain-dependent and distinctive T-cell responses to HIV antigens following immunisation of mice with differing chimpanzee adenovirus vaccine vectors.

In vivo vaccination studies are conventionally conducted in a single mouse strain with results, only reflecting responses to a single immunogenetic background. We decided to examine the immune response to an HIV transgene (gag, pol and nef fusion protein) in 3 strains of mice (CBA, C57BL/6 and BALB/c) to determine the spectrum of responses and in addition to determine whether the serotype of the adenoviral vector used (ChAd3 and ChAd63) impacted the outcome of response. Our results demonstrated that all three strains of mice responded to the transgene and that the magnitude of responses were different between the strains. The C57BL/6 strain showed the lowest range of responses compared to the other strains and, very few responses were seen to the same peptide pool in all three strains of mice. In CBA and BALB/c mice there were significant differences in IFNγ production dependent on the adenoviral vector used. Our results suggest that employing a single strain of mouse may underestimate the efficacy and efficiency of vaccine products.

Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag-pol-nef antigen.

Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene.

The expression of metalloproteinase-2, -9, and -14 and of tissue inhibitors-1 and -2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase-14.

During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and -9 and the membrane gelatinase MMP-14. These, along with the tissue inhibitors TIMP-1 and -2, are developmentally regulated according to the maturation stage of osteoblasts. Their levels change in a similar association with osteoblast phenotypic maturation in different populations of ROB, which take different times to complete osteogenesis in vitro. MMP-14 expression coincides in both cell populations with the mature osteoblastic phenotype and is localized in the cells forming nodules. MMP-2 and -9 are expressed diffusely in the osteoblast population. Developmentally associated changes in the activation of MMP-2 are detected, associated in their timing with the expression of MMP-14 in both populations of ROB, and MMP-14 activates pro-MMP-2 in vitro. Expression of messenger RNAs (mRNAs) for the three MMPs increases up to the time of nodule formation. At this stage, TIMP-1 mRNA levels are lowest. TIMP-2 mRNA decreases throughout osteogenesis. In situ hybridization in 7-day-old rat tibias shows the strongest expression of MMP-14 among osteogenic cells, in lining osteoblasts on the newly formed trabeculae under the growth plate, and on the endosteal surface of cortical bone. Our data support the concept that the developmentally regulated expression of MMP-14 triggers localized proteolysis within the osteogenic population, concomitant in vitro to nodule formation.

A negative search for a paramyxoviral etiology of Paget's disease of bone: molecular, immunological, and ultrastructural studies in UK patients.

Paget's disease of bone is a common bone disease characterized by increased and disorganized bone remodeling at focal sites throughout the skeleton. The etiology of the disease is unresolved. A persistent viral infection has long been suggested to cause the disease. Antigen and/or nucleic acid sequences of paramyxoviruses (in particular measles virus [MV], canine distemper virus [CDV], and respiratory syncytial virus [RSV]) have been reported in pagetic bone by a number of groups; however, others have been unable to confirm this and so far no virus has been isolated from patients. Here, we reexamined the question of viral involvement in Paget's disease in a study involving 53 patients with established disease recruited from seven centers throughout the United Kingdom. Thirty-seven patients showed clear signs of active disease by bone scan and/or histological assessment of the bone biopsy specimens and 12 of these had not received any therapy before samples were taken. Presence of paramyxovirus nucleic acid sequences was sought in bone biopsy specimens, bone marrow, or peripheral blood mononuclear cells using reverse-transcription polymerase chain reaction (RT-PCR) with a total of 18 primer sets (7 of which were nested), including 10 primer sets (including 3 nested sets) specifically for MV or CDV. For each patient at least one sample was tested with all primer sets by RT-PCR and no evidence for the presence of paramyxovirus RNA was found in any patient. In 6 patients, bone biopsy specimens with clear histological evidence of active disease tested negative for presence of measles and CDV using immunocytochemistry (ICC) and in situ hybridization (ISH). Intranuclear inclusion bodies, similar to those described by others previously, were seen in pagetic osteoclasts. The pagetic inclusions were straight, smooth tubular structures packed tightly in parallel bundles and differed from nuclear inclusions, known to represent MV nucleocapsids, in a patient with subacute sclerosing panencephalitis (SSPE) in which undulating, diffuse structures were found, arranged loosely in a nonparallel fashion. In the absence of amplification of viral sequences from tissues that contain frequent nuclear inclusions and given that identical inclusions are found in other bone diseases with a proven genetic, rather than environmental, etiology, it is doubtful whether the inclusions in pagetic osteoclasts indeed represent viral nucleocapsids. Our findings in this large group of patients recruited from throughout the United Kingdom do not support a role for paramyxovirus in the etiology of Paget's disease.

Efficiency of in vivo gene transfer using murine retroviral vectors is strain-dependent in mice.

Retroviral vectors can be used to transduce cultured cells at high frequencies, but efficient transduction of target cells in vivo has proved difficult and little is known about the factors that influence the efficiency of retroviral infection. Many commonly used mouse strains harbor endogenous C-type proviruses, some of which are expressed and have circulating antibodies against the viral envelope glycoproteins that cross-react with the Moloney strain of murine leukemia virus (MoMLV), from which most current retroviral vectors are derived. We have investigated the relative efficiency of retroviral-mediated gene transfer into regenerating skeletal muscle of a variety of mouse strains using a MoMLV-based vector. Humoral immune competence and interference between endogenous MLVs and exogenous recombinant MoMLV were observed to affect the efficiency of retroviral-mediated transfection in vivo. Our results indicate that the mouse genetic background and immune status need to be considered when choosing a preclinical model for in vivo retroviral-mediated gene transfer.

Transplantation of retroviral producer cells for in vivo gene transfer into mouse skeletal muscle.

We describe a new strategy for efficient in vivo gene transfer into skeletal muscle using retroviral vectors. Recombinant retroviral producer cells, previously treated with the cytostatic drug mitomycin C, were injected into regenerating muscle of adult nude, nude/mdx, and C57BL/10 mice. Using LacZ reporter gene activity, we detected efficient transduction in all mouse strains (Nude, mean 11%, range 4.2-21%; C57BL/10, mean 12%, range 3.4-20%; Nude mdx, mean 4.3%, range 2.1-7% at 4 weeks post-injection and 6.6%, range 1.3-12% at 12 weeks post-injection). Foreign gene expression was sustained at high levels for at least 3 months. This strategy allows muscle satellite cells to be transfected in vivo, forming a reservoir of the transgene for incorporation into new myofibers in subsequent rounds of degeneration and regeneration. Because of its efficiency and potentially broad application, this procedure represents a new strategy for in vivo genetic transfer in skeletal muscle and potentially in other tissues.

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

The effect of graft-bed irradiation on the healing of rat skin grafts.

This study explores the possible side effects on healing skin grafts of irradiation, commonly used intraoperatively following surgical tumor removal. The experimental model involved the delivery of a single 10-Gy dose of electron radiation to the recipient bed of a skin wound, followed by attachment of a full thickness rat skin autograft. Skin graft repair was assessed by light microscopy, immunohistochemistry, and transmission electron microscopy over a 3-week period for grafted and grafted-irradiated groups. Graft-bed irradiation reduced fibrinogen, fibrin, and fibronectin deposition in the wound. It also produced brief changes in the extent of both re-epithelialization and granulation tissue formation, and reduced the diameter of collagen fibrils in the granulation tissue. Despite these changes, the results suggest that graft-bed irradiation only delays the healing process, producing no serious clinical complications at the time points studied.

Trans-activation of the murine dystrophin gene in human-mouse hybrid myotubes.

Myotube cultures of the myogenic cell line, C2, produce significantly lower levels of dystrophin than primary mouse cultures. We demonstrate that expression of the C2 dystrophin gene increases 10-fold in hybrid myotubes formed by fusion of C2 and dystrophin-deficient human myoblasts from a Duchenne muscular dystrophy patient. These results indicate that C2 cells are deficient in endogenous gene regulatory factors which enhance dystrophin expression, and that the C2 cell line may therefore be used to identify putative trans-acting factors involved in the regulation of dystrophin gene expression.

Distinct dystrophin mRNA species are expressed in embryonic and adult mouse skeletal muscle.

We have examined dystrophin mRNA in embryonic, newborn and adult mouse skeletal muscle. A discrete nerve-independent increase in mRNA size was observed between embryonic and adult stages, indicating that a developmentally regulated mRNA isoform switch occurs in the expression of the Duchenne muscular dystrophy (DMD) gene in skeletal muscle. These distinct mRNAs are most likely generated via selection of alternative transcriptional start sites or RNA processing pathways. In addition, denervation of adult muscle was without effect on the expression pattern.