As shown in Physique?3A, both the parental Abs and the biAb were still detectable in blood on day 14, albeit at low levels, with a half-life of approximately 30?h (Table S2). defective in binding to several extracellular ligands such as laminin-211 in skeletal muscle tissue, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to numerous symptoms. The genetic heterogeneity associated with the development of -dystroglycanopathies poses significant difficulties to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with -dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an -dystroglycanopathy model, with the biAb improved muscle mass function and guarded muscle tissue from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with -dystroglycanopathy. (like-glycosyltransferase) that leads to a loss in expression, consequent reduced O-linked IIH6-positive matriglycans on DG, and an ability to bind laminin-211 (in a laminin-overlay assay) and thereby render the myofibers susceptible to contraction-induced damage. LARGE is the xylose-glucuronic acid dual glycosyltransferase critical for the addition of the xylose-glucuronic acid disaccharide repeats responsible for ligand binding.15 This reduction in glycosylation was observed in muscle lysates of LARGEmyd-3J mice when compared to wild-type mice using the glycosylation-specific IIH6 antibody (Determine?2A). Both DG and DG were also obvious in the muscle mass lysates of wild-type and LARGEmyd-3J mice, with the latter showing a shift in the size of DG reflecting a loss in glycosylation (Physique?2A). Phenotypically, this mouse model resembles a severe form of LGMD2I or FCMD in humans, with early-onset muscular dystrophy as well as vision, neuromuscular junction, and CNS abnormalities (Physique?S3). When treated with the biAb, the dystroglycan complex was not disrupted as observed in immunoprecipitation assays of muscle mass lysates from mice that had been treated with the biAb, or the parental Abs (Physique?2B). Open in a separate window Physique?2 Localized Intramuscular Injection of the biAb into LARGEmyd-3J Mice Protected TA Muscles Rabbit Polyclonal to PIK3R5 from Exercise-Induced Damage (A) Muscle lysates of non-treated LARGEmyd-3J and wild-type mice show the lack of glycosylation of DG in the LARGE mice by a IIH6-specific antibody and with a polyclonal antibody recognizing both DG and DG. (B) Muscle mass lysate IP from biAb, parental-Ab-, or saline-treated animals shows that biAb treatment does not disrupt the dystroglycan complex as shown by pulldown with the bound SMER18 antibody (left panels) or a DG antibody that has a different epitope than the treatment (right panels; clone 43DAG1/8D5). (C) Summary of EBD-positive fiber counts in biAb- and parental-Ab (control)-treated TA muscle tissue. (D) Representative muscle mass sections of biAb- and parental-Ab-treated TA muscle tissue. Arrows show EBD-positive fibers (orange fibers), and arrowheads represent Ab staining at the sarcolemma (green). Statistical analysis was performed using Students t test; *p? 0.05. n?= 8 animals per group, 6 sections at different planes were assessed per TA. Images SMER18 were taken at 10 magnification. As a proof of concept, LARGEmyd-3J homozygous mice were injected with the biAb into the tibialis anterior (TA) muscle mass; the left TA muscle tissue were injected with the biAb on days 1 and 4, while the right TA muscle tissue were injected with the parental Abdominal muscles as controls. On day 5, Evans blue dye (EBD) was injected intraperitoneally into the mice, and the animals were then subjected to a forced treadmill machine exercise to induce muscle mass damage. As shown in SMER18 Figures 2C and 2D, the EBD-positive fiber counts in the biAb-treated TA muscle tissue were significantly (50%) less than those in the control-treated TA muscle tissue, suggesting that treatment with the biAb conferred a measure of protection against exercise-induced damage. We interpret that this was due to biAb-mediated reconnection of laminin-211 to DG. We also confirmed that administering a mixture of the parental Abdominal muscles to either laminin-211 or DG did not exhibit a dominant-negative effect by conducting the same experiment in wild-type mice (Physique?2C). Systemically Delivered Abdominal muscles Are Localized in the Muscle mass Sarcolemma With protein replacement therapies, biotherapeutics are typically administered to patients via intravenous infusions. Hence, for this concept SMER18 to be efficacious in patients with -dystroglycanopathy, systemic delivery of the biAb needs to result in localization of the agent in a majority of muscle tissues, particularly the sarcolemma, and the biAb needs to be sufficiently stable stability. As shown in Physique?3A, both the parental Abs and the biAb were still detectable in blood on day 14, albeit at low levels, with a half-life of approximately 30?h (Table S2). The parental Abdominal muscles and the biAb were also detected in various muscle mass groups, such as the quadriceps, TA, diaphragm, and heart for up to 14?days (see SMER18 Physique?3B, showing diaphragm and quadriceps as representative examples),.