Human laminin-111 and laminin-211 protein therapy prevents muscle disease progression in an immunodeficient mouse model of LAMA2-CMD

Fig. 1. The NODScid dyW mouse model of LAMA2-CMD shows downregulated levels of laminin-α2 protein chain and is immunodeficient. a Detection of laminin-α2 (red) in tibialis anterior (TA) sections of wild-type, dyW internal control, dyW, and NODScid dyW. Scale bar 100 μm. b Relative levels of immunoglobulin in sera of wild-type, NOD Scid, dyW, and NODScid dyW (N = 9; p value < 0.0001). c Fluorescence-activated cell sorting (FACS) gate analysis of hematopoietic cells (CD45+) from sera of wild-type and NODScid dyW co-labeled with T cell marker (CD3ε+) and B cell marker (CD19+)

Fig. 2. Survival and weight did not change in immunodeficient dyW mice compared to immuno-competent mouse models of LAMA2-CMD. Survival study curves of male (a) and female (b) wild-type, NOD Scid, dyW, and NODScid dyW. c Weekly weights throughout the life span of male and female (d) wild-type, NOD Scid, dyW, and NODScid dyW

Fig. 3. Grip strength is increased in immunodeficient male mice compared to immunocompetent LAMA2-CMD mouse models. Normalized force measurements of grip strength in wild-type, NOD Scid, dyW, and NODScid dyWa male (N = 4, 5, 7, 6, respectively; p value < 0.0001) and b female (N = 5; p value < 0.05*, < 0.0001***)

Fig. 4. Basal regeneration is decreased in immunodeficient compared to immunocompetent mouse models of LAMA2-CMD. a Detection of embryonic Myosin Heavy Chain (eMHC) in TA sections of dyW and NODScid dyW mice. Quantification of eMHC-positive fibers in male (N = 7, 5, respectively; p value of 0.01; scale bar 100 μm) (b) and female (N = 5, 4, respectively; p value of 0.02). c dyW and NODScid dyW mice. Frequency histogram of minimum Feret’s diameters in TA muscle of male (N = 4, 5, respectively; p value < 0.0001) (d) and female (N = 5; p value < 0.0001) (e) dyW and NODScid dyW mice

Fig. 5. Treatment with human recombinant laminin-111 (HsLAM-111) and human recombinant laminin-211 (HsLAM-211) increased muscle regeneration in a mouse model of LAMA2-CMD. a NODScid dyW mice were treated with weekly retro-orbital injections of 1 mg/kg HsLAM-111, HsLAM-211, or PBS from 2 weeks to 6 weeks of age. b Quantification of eMHC-positive fibers in PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dyW (N = 7, 5, 6, respectively; p value < 0.05*, 0.0049**). c Frequency histogram of minimum Feret’s diameters in TA muscle of PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dyW (N = 7, 5, 7, respectively; p value < 0.0001)

Fig. 6. Treatment with HsLAM-111 and HsLAM-211 differentially affects satellite cells and myoblasts in skeletal muscle of LAMA2-CMD. a Quantification of Pax-7-positive satellite cells per frame in TA muscle sections of PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dyW (N = 8, 6, 6, respectively; p value < 0.05* < 0.0001***). Scale bar 100 μm. b Detection of Pax7-positive cells adjacent to fibers in TA sections of NODScid dyW mice. c Quantification of myogenin-positive myoblasts per frame in TA muscle sections of PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dyW (N = 7; p value of 0.019). d Detection of myogenin-positive cells located interstitially in TA sections of NODScid dyW mice

Fig. 7. Treatment with HsLAM-111 improved mouse activity in LAMA2-CMD mouse model. a PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dyW mice pictures and trajectory detected using computer-controlled activity assay. Quantification of distance traveled b, resting time c, and vertical breaks d in PBS-, HsLAM-111-, and HsLAM-211-treated NODScid dyW (N = 7, 7, 6, respectively; p value < 0.05*, < 0.005**)