Compositions and methods for treating muscular dystrophy

Abstract

The present disclosure relates broadly to methods of preventing, reducing the risk of developing, or treating Duchenne muscular dystrophy, Becker muscular dystrophy, Limb-girdle muscular dystrophy (LGMD), Type VI collagen related disorders, congenital muscular dystrophies (CMDs), and congenital myopathies, distal muscular dystrophies / myopathies, comprising administering to a subject an inhibitor of the classical complement pathway, such as a C1 complex inhibitor, a C1 complex inhibitor, a C1q inhibitor, a C1s inhibitor, or a C1r inhibitor.

Description

[Technical field]

[0001] Related Applications This patent application claims priority to U.S. Provisional Patent Application No. 63 / 270,352, filed October 21, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0002] Muscular dystrophies are a group of diseases that make muscles weaker and less flexible over time. Duchenne muscular dystrophy (DMD) is the most common type. It is caused by a defect in the gene that controls how the body keeps muscles healthy.

[0003] Duchenne muscular dystrophy (DMD) is the most common genetic muscle disease in children. Under normal conditions, muscle stem cells (satellite cells) exhibit a very strong regenerative response, but in patients with DMD, the regenerative potential is gradually lost and fibrotic tissue gradually replaces muscle fibers, leading to impaired muscle function.

[0004] Despite the efforts of researchers and medical professionals worldwide trying to address muscular dystrophies, and despite the possibilities offered by genome engineering approaches, there remains a serious need to develop safe and effective treatments for muscular dystrophies, i.e., new therapies are needed to prevent, reduce the risk of developing, and treat muscular dystrophies.

[0005] Muscular dystrophies include a heterogeneous group of genetic disorders characterized by progressive muscle wasting and weakness.In muscular dystrophies, muscle dysfunction results from mutations in genes that code for different cellular components, including proteins associated with sarcolemma, extracellular matrix, nuclear membrane, and sarcolemma organelles.Different forms of dystrophies differ in terms of age of onset, severity of symptomatic progression, and distribution of affected muscles.

[0006] Duchenne muscular dystrophy (DMD, OMIM310200) is one of the most frequent and most severe forms of muscular dystrophy, with an incidence of approximately 1 in 5,000 male newborns. DMD patients usually present in childhood with progressive weakness of limb muscles, trunk muscles, and the diaphragm, eventually leading to wasting, kyphosis, and severe respiratory disease. Becker muscular dystrophy (BMD, OMIM300376) is a rarer (approximately 1 in 20,000 male newborns) and clinically milder form of dystrophy, with the same causative allele as DMD. DMD and BMD are caused by mutations in the DMD gene, which codes for dystrophin.

[0007] Dystrophin is a component of a plasma membrane-associated complex called the dystrophin glycoprotein complex (DGC), which serves as a framework for connecting the intracellular cytoskeleton to the surrounding extracellular matrix. The critical role of the DGC for proper muscle function and integrity is demonstrated by the overlapping pathological features between DMD / BMD and several dystrophies caused by mutations in genes encoding other components of the DGC, namely limb-girdle muscular dystrophies (LGMD) (including sarcoglycanopathies, dystroglycanopathies, and desferlinpathies), collagen VI-related disorders (including Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD)), congenital muscular dystrophies (CMDs) and congenital myopathies, distal muscular dystrophies / myopathies (including Miyoshi myopathy).

[0008] Myotonic dystrophy, facioscapulohumeral dystrophy, Emery-Dreifuss dystrophy, and oculopharyngeal dystrophy appear to have molecular etiologies not attributable to DGC alterations, which is reflected by specific pathological aspects. In particular, myotonic dystrophy (DM) is the most common adult muscular dystrophy and is characterized by autosomal dominant progressive myopathy, muscle tone, and multiorgan involvement. To date, two distinct forms have been identified: myotonic dystrophy type 1 (DM1, Steinert disease), caused by (CTG)n expansions in DMPK, and myotonic dystrophy type 2 (DM2), caused by (CCTG)n expansions in ZNF9 / CNBP. Mutant transcripts aggregate as nuclear foci that sequester RNA-binding proteins, resulting in spliceopathies of downstream effector genes. Summary of the Invention

[0009] The present disclosure broadly relates to a method for preventing, reducing the risk of developing, slowing or blocking the progression of, or treating Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD) (such as sarcoglycanopathies, dystroglycanopathies, and dysferlinopathies), collagen VI-related disorders (such as Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD)), congenital muscular dystrophies (CMD) and congenital myopathies, and distal muscular dystrophies / myopathies (such as Miyoshi myopathy). The method may include administering to a subject an inhibitor of the classical complement pathway, such as a C1 complex inhibitor, a C1q inhibitor, a C1s inhibitor, or a C1r inhibitor. In some aspects, provided herein is a method for preventing, reducing the risk of developing, slowing or blocking the progression of, or treating Duchenne muscular dystrophy. The method can include administering to the subject an inhibitor of the classical complement pathway.

[0010] In some embodiments, the inhibitor of the classical complement pathway is a C1 complex inhibitor, such as an antibody, a peptide, a protein, a nucleic acid, a small molecule, a gene editor, a base editor, or an epigenetic editor. The nucleic acid can be an antisense oligonucleotide, an miRNA, an miRNA inhibitor, an mRNA, an aptamer, or an antisense nucleic acid. The antibody can be an anti-C1 complex antibody, which preferably inhibits C1r or C1s activation or prevents their ability to act on C2 or C4, and / or binds to a combination epitope in the C1 complex, the combination epitope including both C1q and C1s, both C1q and C1r, both C1r and C1s, or each of C1q, C1r, and C1s amino acids.

[0011] In some embodiments, the inhibitor of the classical complement pathway is a C1q inhibitor, such as an antibody, a peptide, a protein, a nucleic acid, a small molecule, a gene editor, a base editor, or an epigenetic editor. The nucleic acid can be an antisense oligonucleotide, an miRNA, an miRNA inhibitor, an mRNA, an aptamer, or an antisense nucleic acid. The antibody can be an anti-C1q antibody, which preferably inhibits the interaction between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s, and / or promotes the clearance of C1q from circulation or tissues. In some embodiments, the anti-C1q antibody has a dissociation constant (KD) in the range of 100 nM to 0.005 nM or less, binds to C1q with a binding stoichiometry in the range of 20:1 to 1.0:1 or less than 1.0:1, and / or binds to C1q with a binding stoichiometry in the range of 6:1 to 1.0:1 or less than 1.0:1 and binds to C1q with a binding stoichiometry in the range of 2.5:1 to 1.0:1 or less than 1.0:1. In some embodiments, the anti-C1q antibody is capable of binding to: (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to IgM, (5) C1q binding to phosphatidylserine, (6) C1q binding to pentraxin-3, (7) C1q binding to C-reactive protein (CRP), (8) C1q binding to globular C1q receptor (gC1qR), (9) C1q binding to complement receptor 1 (CR1), (10) C1q binding to beta-amyloid, (11) C1q binding to calreticulin, (12) C1q binding to amyloid-associated sarcoma, (13) C1q binding to sarcoma, (14) C1q binding to sarcoma, (15) C1q binding to sarcoma, (16) C1q binding to sarcoma, (17) C1q binding to sarcoma, (18) C1q binding to sarcoma, (19) C1q binding to sarcoma, (20) C1q binding to sarcoma, (21) C1q binding to sarcoma, (22) C1q binding to sarcoma, (23) C1q binding to sarcoma, (24) C1q binding to sarcoma, (25) C1q binding to sarcoma, (26) C1q binding to sarcoma, (27) C1q binding to sarcoma, (28) C1q binding to sarcoma, (29) C1q binding to sarcoma, (30) C1q binding to sarcoma, (31) C1q binding to sarcoma, (32) C1q binding to sarcoma, (33) C and / or specifically binds and neutralizes a biological activity of C1q, such as (1) activation of the classical complement activation pathway, (2) activation of antibody and complement dependent cytotoxicity, (3) CH50 hemolysis, (4) synapse loss, (5) B cell antibody production, (6) dendritic cell maturation, (7) T cell proliferation, (8) cytokine production, (9) microglial activation, (10) immune complex formation, (11) phagocytosis of synapses or nerve terminals, (12) activation of complement receptor 3 (CR3 / C3) expressing cells, or (13) neuroinflammation. CH50 hemolysis can include human CH50 hemolysis.In some embodiments, the antibody can neutralize at least about 50% to about 100% of human CH50 hemolysis, and / or the antibody can neutralize at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml, or less than 20 ng / ml. The antibody can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a monovalent antibody, a multispecific antibody, an antibody fragment, or an antibody derivative thereof. In some embodiments, the antibody is an antibody fragment, and the antibody fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single chain antibody molecule.

[0012] In some embodiments, the antibody comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO: 7, and / or a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, the light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO: 7, preferably the light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 4 and 35-38. In some embodiments, the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NO: 8 and 31 to 34, the heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO: 11, preferably the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NO: 8 and 31 to 34. In some embodiments, the antibody is an antibody fragment comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40.

[0013] In some embodiments, the inhibitor of classical complement pathway is a C1r inhibitor, for example, an antibody, a peptide, a protein, a nucleic acid, a small molecule, a gene editing agent, a base editing agent, or an epigenetic editing agent.The nucleic acid is an antisense oligonucleotide, an miRNA, an miRNA inhibitor, an mRNA, an aptamer, or an antisense nucleic acid.In some embodiments, the antibody is an anti-C1r antibody, which preferably inhibits the interaction between C1r and C1q or between C1r and C1s, or the anti-C1r antibody inhibits the catalytic activity of C1r or inhibits the processing of pro-C1r into active protease. In some embodiments, the antibody is an anti-C1r antibody having a dissociation constant (KD) in the range of 100 nM to 0.005 nM or less, the anti-C1r antibody binds to C1r with a binding stoichiometry in the range of 20:1 to 1.0:1 or less, the anti-C1r antibody binds to C1r with a binding stoichiometry in the range of 6:1 to 1.0:1 or less, and / or the anti-C1r antibody binds to C1r with a binding stoichiometry in the range of 2.5:1 to 1.0:1 or less. In some embodiments, the anti-C1r antibody promotes clearance of C1r from circulation or tissues.

[0014] In some embodiments, the inhibitor of classical complement pathway is a C1s inhibitor, for example, an antibody, a peptide, a protein, a nucleic acid, a small molecule, a gene editing agent, a base editing agent, or an epigenetic editing agent.The nucleic acid can be an antisense oligonucleotide, an miRNA, an miRNA inhibitor, an mRNA, an aptamer, or an antisense nucleic acid.In some embodiments, the antibody is an anti-C1s antibody, which preferably inhibits the interaction between C1s and C1q, or between C1s and C1r, or between C1s and C2 or C4, or the anti-C1s antibody inhibits the catalytic activity of C1s, or inhibits the processing of pro-C1s into active protease, or binds to the activated form of C1s. In some embodiments, the anti-C1s antibody has a dissociation constant (KD) in the range of 100 nM to 0.005 nM or less, the anti-C1s antibody binds to C1s with a binding stoichiometry in the range of 20:1 to 1.0:1 or less, the anti-C1s antibody binds to C1s with a binding stoichiometry in the range of 6:1 to 1.0:1 or less, and / or the anti-C1s antibody binds to C1s with a binding stoichiometry in the range of 2.5:1 to 1.0:1 or less. In some embodiments, the anti-C1s antibody promotes clearance of C1s from circulation or tissues.

[0015] In some aspects, provided herein are methods of preventing, reducing the risk of developing, slowing or blocking the progression of, or treating Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD), collagen VI related disorder, congenital muscular dystrophy (CMD) or congenital myopathy, or distal muscular dystrophy / myopathy. The method can include administering to a subject a C1q inhibitory antibody, the antibody comprising a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO:5, HVR-L2 having the amino acid sequence of SEQ ID NO:6, and HVR-L3 having the amino acid sequence of SEQ ID NO:7, and a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO:9, HVR-H2 having the amino acid sequence of SEQ ID NO:10, and HVR-H3 having the amino acid sequence of SEQ ID NO:11. [Brief description of the drawings]

[0016] [Figure 1A] Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1B] Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1C]Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1D] Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1E]Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1F] Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1G]Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1H] Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1I]Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1J] Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1K]Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 1L] Figure 1 shows an assessment of complement protein levels in muscle of 1 year old wild type, 1 year old dystrophic, and 2 year old wild type. ELISA assay of classical complement pathway C1q, C3, and C3d proteins. Tibialis anterior (TA), diaphragm (DIA), and quadriceps (Q) muscles from approximately 1 year old wild type (WT1yo), approximately 1 year old mdx4cv (MDX1yo), and approximately 2 year old wild type (WT2yo) were used. N=3. Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***, and p≦0.0001: ****. [Figure 2A]Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1q protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 2B] Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1q protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 2C]Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1q protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 2D] Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1q protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 2E]Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1q protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 2F] Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1q protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 2G]Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1s protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 2H] Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1s protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 2I]Figure 1 shows that C1q and C1s complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C1s protein in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at approximately 1 month (1 mo), approximately 3 months (3 mo) and 1 year (1 y) (shown on graph). Each sample is normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher23225). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3A] Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C3 protein (shown on graph) of diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3B]Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C3 protein (shown on graph) of diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3C] Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C3 protein (shown on graph) of diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3D]Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C3d protein (shown on graph) in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3E] Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C3d protein (shown on graph) in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3F]Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C3d protein (shown on graph) in diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3G] Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C4 protein (shown on graph) of diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3H]Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C4 protein (shown on graph) of diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 3I] Figure 1 shows that C3, C3d and C4 complement protein levels are increased in dystrophic muscle compared to wild type. ELISA assay of C4 protein (shown on graph) of diaphragm (DIA), tibialis anterior (TA) and quadriceps (Q) muscles of wild type (WT) and mdx4cv (MDX) mice at about 1 month (1 month), about 3 months (3 months) and about 1 year (1 year). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Each sample is normalized to total protein level by dividing by the sample measurement using Pierce™ BCA Protein Assay Kit (ThermoFisher23225). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 4A]Figure 4B-4E shows that dystrophic mice with higher levels of complement proteins compared to wild type are unable to perform physical activity / show muscle wasting. Mouse weights (grams) are shown for wild type (WT) and mdx4Cv (MDX) mice approximately 1 month old (1 mo), approximately 3 months old (3 mo) and approximately 1 year old (1 yo) used in the behavioral tests shown in (Figures 4B-4E). N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1yo). Note that behavioral tests were performed before muscle dissection and ELISA analysis in the same mice as in Figures 2 and 3. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 4B] Figure 1 shows that dystrophic mice with higher levels of complement proteins compared to wild type are unable to perform physical activity / show muscle wasting. Hanging tests performed on wild type (WT) and mdx4Cv (MDX) mice aged approximately 1 month (1 mo), approximately 3 months (3 mo) and approximately 1 year (1 y) are shown. N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1 yo). Note that behavioral tests were performed before muscle dissection and ELISA analysis on the same mice as in Figures 2 and 3. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 4C]Figure 1 shows that dystrophic mice with higher levels of complement proteins compared to wild type are unable to perform physical activity / show muscle wasting. Figure 2 shows the open field test performed on wild type (WT) and mdx4Cv (MDX) mice aged approximately 1 month (1 mo), approximately 3 months (3 mo) and approximately 1 year (1 y). Total distance (m) was evaluated for each group of animals. N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1 yo). Note that behavioral testing was performed before muscle dissection and ELISA analysis in the same mice as in Figures 2 and 3. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 4D] Figure 1 shows that dystrophic mice with higher levels of complement proteins compared to wild type are unable to perform physical activity / show muscle wasting. Figure 2 shows open field tests performed on wild type (WT) and mdx4Cv (MDX) mice aged approximately 1 month (1 mo), approximately 3 months (3 mo) and approximately 1 year (1 y). For each group of animals, the average speed (m / sec) was evaluated. N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1 yo). Note that behavioral tests were performed before muscle dissection and ELISA analysis in the same mice as in Figures 2 and 3. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 4E]Figure 1 shows that dystrophic mice with higher levels of complement proteins compared to wild type are unable to perform physical activity / show muscle wasting. Figure 2 shows an open field test performed on wild type (WT) and mdx4Cv (MDX) mice aged approximately 1 month (1 mo), approximately 3 months (3 mo) and approximately 1 year (1 y). For each group of animals, movement time (sec) was evaluated. N=3 (WT 1 mo, WT 3 mo), N=4 (WT 1 yo, MDX 1 mo, MDX 3 mo, MDX 1 yo). Note that behavioral testing was performed before muscle dissection and ELISA analysis in the same mice as in Figures 2 and 3. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Diagram 5] Experimental design of anti-C1q treatment of dystrophic mice is shown. C1q antibody treatment was administered in vivo to Pax7CreER;R26RYFP;mdx4Cv male mice for 2 weeks at a regimen of 100 mg / kg intraperitoneally (ip) twice per week starting at 10 weeks of age. Blood samples were taken at the start of pharmacological treatment and at sacrifice. Functional parameters (i.e., maximum hanging time before fatigue, and behavioral activity such as total distance and average speed run) were assessed at the start and end of treatment. [Figure 6A] Validation of C1qaKO;mdx4Cv mice. qPCR analysis of C1qa expression in FACS-isolated macrophages (CD45+F4 / 80+) from hindlimb muscles of approximately 3-month-old LyzCre+ / -C1qaFL / FL;mdx4Cv (C1qaKO, N=4) and approximately 3-11-month-old LyzCre+ / -C1qaWT / WT;mdx4Cv (Cntr, N=7). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 6B]Validation of C1qaKO;mdx4Cv mice. qPCR analysis of C1qa expression in diaphragm and gastrocnemius muscles of LyzCre+ / -C1qaWT / WT;mdx4Cv (Cntr(Cre+), N=6), LyzCre+ / -C1qaFL / FL;mdx4Cv (C1qaKO, N=5), and wild type (WT, N=3) mice at approximately 3 months of age. Data are expressed as mean with SEM. One-way anova test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 6C] Validation of C1qaKO;mdx4Cv mice. qPCR analysis of C1qa expression in diaphragm and gastrocnemius muscles of LyzCre+ / -C1qaWT / WT;mdx4Cv (Cntr(Cre+), N=6), LyzCre+ / -C1qaFL / FL;mdx4Cv (C1qaKO, N=5), and wild type (WT, N=3) mice at approximately 3 months of age. Data are expressed as mean with SEM. One-way anova test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 7A] Behavioral testing in 1-month-old C1qaKO;mdx4Cv mice and controls. Mouse weights (grams) are shown for LyzCre+ / -C1qaFL / FL;mdx4Cv (C1qaKO) and LyzCre+ / -C1qaWT / WT;mdx4Cv (CNTR) mice at approximately 1 month of age. N=9 (C1qaKO), N=8 (CNTR). Tests were repeated 3 times, every other day. The average values ​​for the 3 days for each test are shown. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 7B]Behavioral testing in 1-month-old C1qaKO;mdx4Cv mice and controls. Hanging test (HT) performed on mice as in (Figure 7A) is shown. Total hanging time was assessed. N=9 (C1qaKO), N=8 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 7C] Behavioral testing in 1-month-old C1qaKO;mdx4Cv mice and controls. Hanging test (HT) performed on mice as in (Figure 7A) is shown. Total hanging time normalized to mouse weight was assessed. N=9 (C1qaKO), N=8 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 7D] Behavioral testing in 1-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) test performed on mice as in (Figure 7A). Total distance (cm) was assessed. N=9 (C1qaKO), N=8 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 7E]Behavioral testing in 1-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) testing performed on mice as in (Figure 7A) is shown. Mean speed (cm / sec) was assessed. N=9 (C1qaKO), N=8 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 7F] Behavioral testing in 1-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) test performed on mice as in (Figure 7A). Percentage of movement time was assessed. N=9 (C1qaKO), N=8 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 8A] Behavioral testing in 2-month-old C1qaKO;mdx4Cv mice and controls. Mouse weights (grams) are shown for LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO) and LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR) mice at approximately 2 months of age. N=4. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 8B] Behavioral testing in 2-month-old C1qaKO;mdx4Cv mice and controls. Hanging test (HT) performed on mice as in (Figure 8A) is shown. Total hanging time was assessed. N=4. Data are presented as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 8C]Behavioral testing in 2-month-old C1qaKO;mdx4Cv mice and controls. Hanging test (HT) performed on mice as in (Figure 8A) is shown. Total hanging time normalized to mouse weight was assessed. N=4. Data are presented as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 8D] Behavioral testing in 2-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) test performed on mice as in (Figure 8A). Total distance (cm) was assessed. N=4. Data are presented as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 8E] Behavioral testing in 2-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) testing performed on mice as in (Figure 8A) is shown. Mean velocity (cm / sec) was assessed. N=4. Data are presented as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 8F] Behavioral testing in 2-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) test performed on mice as in (Figure 8A). Percentage of movement time was assessed. N=4. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 9A]Behavioral testing in 3-month-old C1qaKO;mdx4Cv mice and controls. Mouse weights (grams) are shown for LyzCre+ / -C1qaFL / FL;mdx4Cv (C1qaKO) and LyzCre+ / -C1qaWT / WT;mdx4Cv (CNTR) mice at approximately 3 months of age. N=9 (C1qaKO), N=7 (CNTR). Tests were repeated 3 times, every other day. The average values ​​for the 3 days for each test are shown. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 9B] Behavioral testing in 3-month-old C1qaKO;mdx4Cv mice and controls. Hanging test (HT) performed on mice as in (Figure 9A) is shown. Total hanging time was assessed. N=9 (C1qaKO), N=7 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 9C] Behavioral testing in 3-month-old C1qaKO;mdx4Cv mice and controls. Hanging test (HT) performed on mice as in (Figure 9A) is shown. Total hanging time normalized to mouse weight was assessed. N=9 (C1qaKO), N=7 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 9D]Behavioral testing in 3-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) test performed on mice as in (Figure 9A). Total distance (cm) was assessed. N=9 (C1qaKO), N=7 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 9E] Behavioral testing in 3-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) testing performed on mice as in (Figure 9A) is shown. Mean speed (cm / sec) was assessed. N=9 (C1qaKO), N=7 (CNTR). Tests were repeated 3 times, every other day. The average values ​​of the 3 days for each test are shown. Data are expressed as mean with SEM. Two-tailed unpaired t-tests were applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 9F] Behavioral testing in 3-month-old C1qaKO;mdx4Cv mice and controls. Open field (OF) test performed on mice as in (Figure 9A). Percentage of movement time was assessed. N=9 (C1qaKO), N=7 (CNTR). Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 10A]Behavioral testing in 3-month-old dystrophic mice treated with anti-C1q antibody. Mouse weights (grams) are shown for Pax7CreER;R26RYFP;mdx4Cv mice treated with anti-C1q blocking antibody (anti-C1q) or control antibody (Cntr) before (pre) and after (post) treatment. N=6. Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ***. [Figure 10B] Behavioral testing in dystrophic mice aged 3 months treated with anti-C1q antibody. Hanging test (HT) performed on mice as in (Figure 10A). Total hanging time was evaluated. N=6. Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ***. [Figure 10C] Behavioral testing in dystrophic mice aged 3 months treated with anti-C1q antibody. Hanging test (HT) performed on mice as in (Figure 10A) is shown. Total hanging time normalized for mouse weight was evaluated. N=6. Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ***. [Figure 10D] Behavioral testing in dystrophic mice aged 3 months treated with anti-C1q antibody. Open field (OF) test performed on mice as in (Figure 10A). Total distance (cm) was assessed. N=6. Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ***. [Figure 10E] Behavioral testing in dystrophic mice aged 3 months treated with anti-C1q antibody. Open field (OF) test performed on mice as in (Figure 10A). Mean speed (cm / sec) was assessed. N=6. Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ***. [Figure 10F] Behavioral testing in dystrophic mice aged 3 months treated with anti-C1q antibody. Open field (OF) test performed on mice as in (Figure 10A). Percentage of movement time was assessed. N=6. Tests were repeated 3 times, every other day. The average value of the 3 days for each test is shown. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ***. [Figure 11A] Canonical Wnt signaling and fibrogenicity-related gene expression in the diaphragm of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Tgfβ gene in the diaphragm of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 11B]Canonical Wnt signaling and fibrogenicity-related gene expression in the diaphragm of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Lgr5 gene in the diaphragm of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age, treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 11C] Canonical Wnt signaling and fibrogenicity-related gene expression in the diaphragm of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of collagen 1a1 gene in the diaphragm of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 11D]Canonical Wnt signaling and fibrogenic-related gene expression in the diaphragm of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of collagen 3a1 gene in the diaphragm of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 11E] Canonical Wnt signaling and fibrogenicity-related gene expression in the diaphragm of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of fibronectin gene in the diaphragm of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age, treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 12A]Canonical Wnt signaling and fibrogenic-related gene expression in gastrocnemius muscle of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Tgfβ gene in gastrocnemius muscle of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 12B] Canonical Wnt signaling and fibrogenic-related gene expression in gastrocnemius muscle of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Lgr5 gene in gastrocnemius muscle of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 12C]Canonical Wnt signaling and fibrogenic-related gene expression in gastrocnemius muscle of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of collagen 1a1 gene in gastrocnemius muscle of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 12D] Canonical Wnt signaling and fibrogenic-related gene expression in gastrocnemius muscle of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of collagen 3a1 gene in gastrocnemius muscle of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 12E]Canonical Wnt signaling and fibrogenicity-related gene expression in gastrocnemius muscle of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of fibronectin gene in gastrocnemius muscle of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 13A] Canonical Wnt signaling and fibrogenic-related gene expression in fibro / adipogenic progenitor cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Tgfβ gene in fibro / adipogenic progenitor cells from LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 13B]Canonical Wnt signaling and fibrogenic-related gene expression in fibro / adipogenic progenitor cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Lgr5 gene in fibro / adipogenic progenitor cells from LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 13C] Canonical Wnt signaling and fibrogenic-related gene expression in fibro / adipogenic progenitor cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Axin2 gene in fibro / adipogenic progenitor cells from LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 13D]Canonical Wnt signaling and fibrogenic-related gene expression in fibro / adipogenic progenitor cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of collagen 1a1 gene in fibro / adipogenic progenitor cells from LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 13E] Canonical Wnt signaling and fibrogenic-related gene expression in fibro / adipogenic progenitor cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of collagen 3a1 gene in fibro / adipogenic progenitor cells from LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 13F]Canonical Wnt signaling and fibrogenic-related gene expression in fibro / adipogenic progenitor cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of fibronectin gene in fibro / adipogenic progenitor cells from LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age and treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 14A] Expression of canonical Wnt signaling and fibrogenicity-related genes in satellite cells of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Tgfβ gene in satellite cells of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age, treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 14B]Expression of canonical Wnt signaling and fibrogenicity-related genes in satellite cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Lgr5 gene in satellite cells from approximately 3-month-old LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild-type (WT), Pax7CreER;R26RYFP;mdx4Cv mice treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 14C] Expression of canonical Wnt signaling and fibrogenicity-related genes in satellite cells of C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of Axin2 gene in satellite cells of LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice at approximately 3 months of age, treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 14D]Expression of canonical Wnt signaling and fibrogenicity-related genes in satellite cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of collagen 1a1 gene in satellite cells from approximately 3-month-old LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild-type (WT), Pax7CreER;R26RYFP;mdx4Cv mice treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 14E] Expression of canonical Wnt signaling and fibrogenicity-related genes in satellite cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody. qPCR analysis of collagen 3a1 gene in satellite cells from approximately 3-month-old LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 14F]Expression of canonical Wnt signaling and fibrogenicity-related genes in satellite cells from C1qaKO;mdx4Cv mice and dystrophic mice treated with anti-C1q antibody.qPCR analysis of fibronectin gene in satellite cells from approximately 3-month-old LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)), wild type (WT), Pax7CreER;R26RYFP;mdx4Cv mice treated with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 15A] Creatine kinase assay in serum from dystrophic C1qaKO and dystrophic mice treated with anti-C1q antibody. CK activity (nmol / min / mL) measured in serum samples taken from approximately 3-month-old LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO(Cre+)), LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR(Cre+)) and wild type (WT). N=6 (CNTR(Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. One-way ANOVA test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 15B]Creatine kinase assay in serum from dystrophic C1qaKO and dystrophic mice treated with anti-C1q antibody. CK activity (nmol / min / mL) measured in serum samples taken from Pax7CreER;R26RYFP;mdx4Cv mice before (pre) and after (post) treatment with C1q blocking antibody (anti-C1q) or control antibody (Cntr). N=6 (CNTR (Cre+), N=5 (C1qaKO, Cntr, anti-C1q), N=3 (WT). Data are expressed as mean with SEM. One-way ANOVA test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 16A] Figure 1 shows an assessment of complement levels in plasma collected from C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), and C1s-C1 inhibitor complex (C1sC1inh). PK indicates the amount of C1q blocking antibody in the sample. Plasma samples were taken from the following animals: mice treated with anti-C1q blocking antibody (indicated as B in the graph) before (pre) and after (post) treatment, mice treated with control antibody (indicated as A in the graph) before (pre) and after (post) treatment, LyzCre+ / -C1qaFL / FL; mdx4Cv (C1qaKO Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre+ / -C1qaWT / WT; mdx4Cv (Cntr Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre- / -C1qaFL / FL; mdx4Cv or LyzCre- / -C1qaWT / FL; mdx4Cv (Cntr Cre-) mice at 1 month and sacrifice (3 months of age), and wild type (WT) mice at 3 months of age. N=5 (A, B, C1qaKO Cre+), N=6 (Cntr Cre+), N=4 (Cntr Cre-), N=3 (WT). Data are expressed as mean with SEM. [Figure 16B]Figure 1 shows an assessment of complement levels in plasma collected from C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), and C1s-C1 inhibitor complex (C1sC1inh). PK indicates the amount of C1q blocking antibody in the sample. Plasma samples were taken from the following animals: mice treated with anti-C1q blocking antibody (indicated as B in the graph) before (pre) and after (post) treatment, mice treated with control antibody (indicated as A in the graph) before (pre) and after (post) treatment, LyzCre+ / -C1qaFL / FL; mdx4Cv (C1qaKO Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre+ / -C1qaWT / WT; mdx4Cv (Cntr Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre- / -C1qaFL / FL; mdx4Cv or LyzCre- / -C1qaWT / FL; mdx4Cv (Cntr Cre-) mice at 1 month and sacrifice (3 months of age), and wild type (WT) mice at 3 months of age. N=5 (A, B, C1qaKO Cre+), N=6 (Cntr Cre+), N=4 (Cntr Cre-), N=3 (WT). Data are expressed as mean with SEM. [Figure 16C]Figure 1 shows an assessment of complement levels in plasma collected from C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), and C1s-C1 inhibitor complex (C1sC1inh). PK indicates the amount of C1q blocking antibody in the sample. Plasma samples were taken from the following animals: mice treated with anti-C1q blocking antibody (indicated as B in the graph) before (pre) and after (post) treatment, mice treated with control antibody (indicated as A in the graph) before (pre) and after (post) treatment, LyzCre+ / -C1qaFL / FL; mdx4Cv (C1qaKO Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre+ / -C1qaWT / WT; mdx4Cv (Cntr Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre- / -C1qaFL / FL; mdx4Cv or LyzCre- / -C1qaWT / FL; mdx4Cv (Cntr Cre-) mice at 1 month and sacrifice (3 months of age), and wild type (WT) mice at 3 months of age. N=5 (A, B, C1qaKO Cre+), N=6 (Cntr Cre+), N=4 (Cntr Cre-), N=3 (WT). Data are expressed as mean with SEM. [Figure 16D]Figure 1 shows an assessment of complement levels in plasma collected from C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), and C1s-C1 inhibitor complex (C1sC1inh). PK indicates the amount of C1q blocking antibody in the sample. Plasma samples were taken from the following animals: mice treated with anti-C1q blocking antibody (indicated as B in the graph) before (pre) and after (post) treatment, mice treated with control antibody (indicated as A in the graph) before (pre) and after (post) treatment, LyzCre+ / -C1qaFL / FL; mdx4Cv (C1qaKO Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre+ / -C1qaWT / WT; mdx4Cv (Cntr Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre- / -C1qaFL / FL; mdx4Cv or LyzCre- / -C1qaWT / FL; mdx4Cv (Cntr Cre-) mice at 1 month and sacrifice (3 months of age), and wild type (WT) mice at 3 months of age. N=5 (A, B, C1qaKO Cre+), N=6 (Cntr Cre+), N=4 (Cntr Cre-), N=3 (WT). Data are expressed as mean with SEM. [Figure 16E]Figure 1 shows an assessment of complement levels in plasma collected from C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), and C1s-C1 inhibitor complex (C1sC1inh). PK indicates the amount of C1q blocking antibody in the sample. Plasma samples were taken from the following animals: mice treated with anti-C1q blocking antibody (indicated as B in the graph) before (pre) and after (post) treatment, mice treated with control antibody (indicated as A in the graph) before (pre) and after (post) treatment, LyzCre+ / -C1qaFL / FL; mdx4Cv (C1qaKO Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre+ / -C1qaWT / WT; mdx4Cv (Cntr Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre- / -C1qaFL / FL; mdx4Cv or LyzCre- / -C1qaWT / FL; mdx4Cv (Cntr Cre-) mice at 1 month and sacrifice (3 months of age), and wild type (WT) mice at 3 months of age. N=5 (A, B, C1qaKO Cre+), N=6 (Cntr Cre+), N=4 (Cntr Cre-), N=3 (WT). Data are expressed as mean with SEM. [Figure 16F]Figure 1 shows an assessment of complement levels in plasma collected from C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), and C1s-C1 inhibitor complex (C1sC1inh). PK indicates the amount of C1q blocking antibody in the sample. Plasma samples were taken from the following animals: mice treated with anti-C1q blocking antibody (indicated as B in the graph) before (pre) and after (post) treatment, mice treated with control antibody (indicated as A in the graph) before (pre) and after (post) treatment, LyzCre+ / -C1qaFL / FL; mdx4Cv (C1qaKO Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre+ / -C1qaWT / WT; mdx4Cv (Cntr Cre+) mice at 1 month and sacrifice (3 months of age), LyzCre- / -C1qaFL / FL; mdx4Cv or LyzCre- / -C1qaWT / FL; mdx4Cv (Cntr Cre-) mice at 1 month and sacrifice (3 months of age), and wild type (WT) mice at 3 months of age. N=5 (A, B, C1qaKO Cre+), N=6 (Cntr Cre+), N=4 (Cntr Cre-), N=3 (WT). Data are expressed as mean with SEM. [Figure 17A]Complement level assessment in the diaphragm of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Data are presented as mean with SEM. [Figure 17B]Complement level assessment in the diaphragm of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Data are presented as mean with SEM. [Figure 17C]Complement level assessment in the diaphragm of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Data are presented as mean with SEM. [Figure 17D]Complement level assessment in the diaphragm of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Data are presented as mean with SEM. [Figure 17E]Complement level assessment in the diaphragm of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Data are presented as mean with SEM. [Figure 17F]Complement level assessment in the diaphragm of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Data are presented as mean with SEM. [Figure 17G]Complement level assessment in the diaphragm of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Data are presented as mean with SEM. [Figure 18A]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 18B]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 18C]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 18D]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 18E]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 18F]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 18G]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 18H]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 18I]Complement level assessment in diaphragms of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), diaphragms were collected from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=4 (A, B, C1qaKO Cre+, Cntr Cre+), N=3 (Cntr Cre-, WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 19A]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody. ELISA assays of proteins of classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are presented as mean with SEM. [Figure 19B]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody. ELISA assays of proteins of classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are presented as mean with SEM. [Figure 19C]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody. ELISA assays of proteins of classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are presented as mean with SEM. [Figure 19D]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody. ELISA assays of proteins of classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are presented as mean with SEM. [Figure 19E]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody. ELISA assays of proteins of classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are presented as mean with SEM. [Figure 19F]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody. ELISA assays of proteins of classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are presented as mean with SEM. [Figure 19G]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody. ELISA assays of proteins of classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are presented as mean with SEM. [Figure 20A]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20B]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20C]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20D]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20E]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20F]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20G]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20H]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20I]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20J]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20K]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20L]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 20M]Complement level assessment in gastrocnemius muscle of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibody (total protein corrected). ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. All data are expressed as protein (or protein complex) / total protein ratio. At the time of sacrifice (3 months of age), gastrocnemius muscles were harvested from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Each sample was normalized to total protein levels by dividing by the sample measurement using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225). Data are expressed as mean with SEM. [Figure 21A]Complement level assessment in livers of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), livers were taken from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are expressed as mean with SEM. [Figure 21B]Complement level assessment in livers of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), livers were taken from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are expressed as mean with SEM. [Figure 21C]Complement level assessment in livers of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), livers were taken from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are expressed as mean with SEM. [Figure 21D]Complement level assessment in livers of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), livers were taken from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are expressed as mean with SEM. [Figure 21E]Complement level assessment in livers of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), livers were taken from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are expressed as mean with SEM. [Figure 21F]Complement level assessment in livers of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), livers were taken from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are expressed as mean with SEM. [Figure 21G]Complement level assessment in livers of C1qaKO;mdx4Cv and dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d, and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh), and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. At the time of sacrifice (3 months of age), livers were taken from the following animals: mice treated with anti-C1q blocking antibody (shown as B in the graph), mice treated with control antibody (shown as A in the graph), LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO Cre+) mice, LyzCre+ / -C1qaWT / WT;mdx4Cv(Cntr Cre+) mice, LyzCre- / -C1qaFL / FL;mdx4Cv or LyzCre- / -C1qaWT / FL;mdx4Cv(Cntr Cre-) mice, and wild type (WT). N=5 (A, B, C1qaKO Cre+), N=4 (Cntr Cre-), N=6 (Cntr Cre+), N=3 (WT). Data are expressed as mean with SEM. [Figure 22A] Figure 1 shows the assessment of complement levels in the hearts of dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. Hearts were taken at sacrifice (3 months of age) from mice treated with anti-C1q blocking antibody (shown as B on the graph) and from mice treated with control antibody (shown as A on the graph); N=5. Data are presented as mean with SEM. [Figure 22B]Figure 1 shows the assessment of complement levels in the hearts of dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. Hearts were taken at sacrifice (3 months of age) from mice treated with anti-C1q blocking antibody (shown as B on the graph) and from mice treated with control antibody (shown as A on the graph); N=5. Data are presented as mean with SEM. [Figure 22C] Figure 1 shows the assessment of complement levels in the hearts of dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. Hearts were taken at sacrifice (3 months of age) from mice treated with anti-C1q blocking antibody (shown as B on the graph) and from mice treated with control antibody (shown as A on the graph); N=5. Data are presented as mean with SEM. [Figure 22D] Figure 1 shows the assessment of complement levels in the hearts of dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. Hearts were taken at sacrifice (3 months of age) from mice treated with anti-C1q blocking antibody (shown as B on the graph) and from mice treated with control antibody (shown as A on the graph); N=5. Data are presented as mean with SEM. [Figure 22E]Figure 1 shows the assessment of complement levels in the hearts of dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. Hearts were taken at sacrifice (3 months of age) from mice treated with anti-C1q blocking antibody (shown as B on the graph) and from mice treated with control antibody (shown as A on the graph); N=5. Data are presented as mean with SEM. [Figure 22F] Figure 1 shows the assessment of complement levels in the hearts of dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. Hearts were taken at sacrifice (3 months of age) from mice treated with anti-C1q blocking antibody (shown as B on the graph) and from mice treated with control antibody (shown as A on the graph); N=5. Data are presented as mean with SEM. [Figure 22G] Figure 1 shows the assessment of complement levels in the hearts of dystrophic mice treated with anti-C1q antibodies. ELISA assays of proteins of the classical complement pathway (i.e., C1q, C3d and C1s), C1q-C3d immune complex (IC), C1s-C1 inhibitor complex (C1sC1inh) and albumin (Alb). PK indicates the amount of C1q blocking antibody in the sample. Hearts were taken at sacrifice (3 months of age) from mice treated with anti-C1q blocking antibody (shown as B on the graph) and from mice treated with control antibody (shown as A on the graph); N=5. Data are presented as mean with SEM. [Figure 23]Figure 1 shows that expression of C1 complex components is enhanced in dystrophic muscle. mRNA expression of C1 complex subunits C1qa, C1qb, C1qc, C1r, C1s in hindlimb muscles of wild type (WT) and mdxCv (Mdx) mice at approximately 1 year of age. N=3. Data are presented as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 24A] Figure 1 shows that C1q subunits are expressed by infiltrating macrophages in skeletal muscle of dystrophic mice. qPCR analysis of C1qa expression in satellite cells (SCs), macrophages (MACs) and fibrotic / adipogenic progenitor cells (FAPs) isolated from hindlimb muscles of wild type (WT) and mdx4Cv (Mdx) mice at approximately 1 year of age. N=3. Data are presented as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 24B] Figure 1 shows that C1q subunits are expressed by infiltrating macrophages in skeletal muscle of dystrophic mice. qPCR analysis of C1qb expression in satellite cells (SCs), macrophages (MACs) and fibrotic / adipogenic progenitor cells (FAPs) isolated from hindlimb muscles of wild type (WT) and mdx4Cv (Mdx) mice at approximately 1 year of age. N=3. Data are presented as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 24C] Figure 1 shows that the C1q subunit is expressed by infiltrating macrophages in skeletal muscle of dystrophic mice. The number of macrophages per mg of tissue in hindlimb muscle of wild type (WT) and mdx4Cv (Mdx) mice at approximately 1 year of age is shown. N=3. Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: ***; p≦0.0001: ****. [Figure 25A] Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age is shown. Mouse weights (grams) are shown for LyzCre+ / -C1qaFL / FL;mdx4Cv(C1qaKO) and LyzCre+ / -C1qaWT / WT;mdx4Cv(CNTR) at approximately 1 year of age. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 25B] Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age. Hanging test (HT) performed on mice as in (Figure 25A) is shown. Total hanging time was assessed. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 25C] Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age. Hanging test (HT) performed on mice as in (Figure 25A) is shown. Total hanging time normalized to mouse weight was assessed. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 25D] Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age. Open field (OF) testing performed on mice as in (Figure 25A) is shown. Total distance (cm) was assessed. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 25E]Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age. Open field (OF) testing performed on mice as in (FIG. 25A) is shown. Mean speed (cm / sec) was assessed. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 25F] Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age. Open field (OF) test performed on mice as in (Figure 25A) is shown. Percentage of movement time was assessed. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 25G] Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age. Two-leg grip test performed on mice as in (FIG. 25A). Maximal strength normalized to mouse weight was assessed. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Fig. 25H] Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age. Two-leg grip test performed on mice as in (FIG. 25A) is shown. Total grip time normalized to mouse weight was assessed. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 25I]Behavioral testing in C1qaKO;mdx4Cv mice and controls at approximately 1 year of age. Rotarod test performed on mice as in (FIG. 25A). Total walking time normalized to mouse weight was assessed. N=5 (C1qaKO), N=7 (Cntr), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****. [Figure 26] Figure 1 shows that expression of C1 complex components was enhanced in dystrophic muscle. mRNA expression of C1 complex subunits C1qa, C1qb, C1qc, C1r, C1s was assessed in hindlimb muscles of approximately 1-year-old wild-type (WT) and mdx4Cv (MDX) mice (N=3). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001. [Figure 27] Representative immunofluorescence of gastrocnemius muscle of approximately 1 year old mdx4Cv stained with anti-C1q, anti-Axin2 and Hoechst. Scale bar (upper image): 50 μm, Scale bar (lower image): 10 μm. Positive correlation between C1q and Axin2 intensity values ​​in each region is shown. N=3. Spearman coefficient: r. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] overview The present disclosure relates broadly to methods of preventing, reducing the risk of developing, slowing or blocking the progression of, or treating Duchenne muscular dystrophy ("DMD"), Becker muscular dystrophy ("BMD"), limb-girdle muscular dystrophies (LGMD) (including sarcoglycanopathies, dystroglycanopathies, and dysferlinopathies), collagen VI related disorders (including Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD)), congenital muscular dystrophies (CMDs) and congenital myopathies, and distal muscular dystrophies / myopathies (including Miyoshi myopathy), comprising administering to a subject an inhibitor of the classical complement pathway, such as a C1 complex inhibitor, a C1q inhibitor, a C1s inhibitor, a C1r inhibitor, or a C1 complex inhibitor (e.g., an anti-C1 complex antibody). The inhibitor can be an antibody, a peptide, a protein, a nucleic acid, a small molecule, a gene editing agent, a base editing agent, or an epigenetic editing agent.The nucleic acid can be an antisense oligonucleotide, an miRNA, an miRNA inhibitor, an mRNA, an aptamer, or an antisense nucleic acid.The inhibitor can refer to a compound that has the ability to inhibit the biological function of the target biomolecule, whether by reducing the activity or expression of the target biomolecule.

[0018] The complement system is involved in the necrotic process that occurs in muscle fibers during the progression of DMD, and the alternative complement pathway may be directly involved in tissue regeneration. However, the role of the classical complement pathway in the progression of DMD is poorly understood, and to date its involvement in muscle regeneration is unknown. Although changes in serum levels of complement proteins have been reported in compromised skeletal muscles (i.e., aging and muscle diseases), the local production of complement components in skeletal muscles has not been extensively investigated in either physiological or pathological conditions.

[0019] Elevated WNT signaling has been shown to play a detrimental role in regeneration processes and promote the accumulation of fibrotic tissue in dystrophic muscle. However, the molecular and cellular pathways involved in this process are poorly characterized. In addition to its role in innate immunity, classical complement component C1q can activate canonical WNT signaling. In some embodiments, the disclosed method of treating muscular dystrophy is based in part on the discovery that complement C1q correlates with enhanced activity of the WNT signaling pathway in DMD (Figure 27).

[0020] C1q-Wnt inhibition effectively ameliorates the dystrophic phenotype in a mouse model of Duchenne muscular dystrophy in vivo.

[0021] Complement C1 complex RNA and protein levels are increased 10-fold as early as the first month of life and are more abundant in dystrophic mdx mice compared with healthy controls. 4Cv It remains elevated in skeletal muscle up to 1 year of age. The anti-C1q blocking antibody regimen used in the study described in the Examples herein effectively inhibited C1q expression in target skeletal muscles (i.e., diaphragm and gastrocnemius). Dystrophic mice treated with anti-C1q blocking antibodies had increased maximum hanging time before fatigue compared to dystrophic mice treated with control antibodies. Dystrophic mice genetically ablated C1qa (i.e., C1qa KO ;mdx 4Cv A similar trend suggesting improved maximum hanging time before fatigue was observed in mice (12.5- and 15.1-month-olds) compared to controls tested at approximately 1 and 2 months of age. This improvement was not evident after 1 month, suggesting a possible transient effect exerted by genetic ablation of C1q. KO ;mdx 4Cv The rats resisted the grip test for more time compared to controls and showed a trend toward increased maximum hanging time before fatigue compared to controls. Overall, gene expression of canonical WNT target genes and fibrogenic genes was significantly increased by C1qa KO ;mdx 4CvThe C1qa expression level was not decreased in dystrophic mice treated with anti-C1q blocking antibodies, nor in dystrophic mice treated with anti-C1q blocking antibodies. KO ;mdx 4Cv A trend suggesting a decrease in serum creatine kinase (CK) activity was observed, however, no difference in CK activity levels was observed in dystrophic mice treated with anti-C1q antibody compared to controls.

[0022] First, complement levels, specifically the expression of classical complement pathway components, were assessed in muscle of dystrophic and wild-type mice, and complement protein levels were increased as early as one month of age and were significantly increased in dystrophic mdx mice compared to healthy controls. 4Cv It was found to remain elevated in muscle up to 1 year of age.

[0023] ELISA analysis performed on tissues (i.e., diaphragm, gastrocnemius, and liver) taken from dystrophic mice treated with anti-C1q blocking antibodies demonstrated that the regimen employed reduced the expression of C1q compared with C1qa genetically ablated mouse tissues (i.e., C1qa KO ;mdx 4Cv ) to a level similar to that in the control. CreER ;R26R YFP ;mdx 4Cv Treating mice with anti-C1q blocking antibody intraperitoneally (ip) for 2 weeks starting at 10 weeks of age at a dosage of 100 mg / kg on a regimen of 2 times per week is an effective method to deplete C1q skeletal muscle and liver expression.

[0024] Having assessed that the anti-C1q blocking antibody regimen effectively depleted C1q expression in dystrophic skeletal muscle, we assessed the effect of C1q depletion on mouse functional parameters, gene expression of canonical WNT target genes and fibrogenicity-related genes, and creatine kinase activity levels.

[0025] At both approximately 1 and 2 months of age, C1qa was significantly increased in the four-limb hanging wire test compared to controls. KO ;mdx 4Cv We observed improved physical activity in mice treated with anti-C1q antibodies. However, this trend was only transient and was no longer observed when the mice were tested at approximately 3 months of age. Dystrophic mice treated with anti-C1q antibodies produced higher physical activity in the four-limb hanging wire test compared to mice treated with a control antibody.

[0026] definition As used herein, "a" or "an" can mean one or more. As used herein in the claim(s), when used in conjunction with the term "comprising", the term "a" or "an" can mean one or more. For example, a reference to an "antibody" is a reference to one to many antibodies. As used herein, "another" can mean at least a second or more.

[0027] As used herein, administration "in conjunction with" another compound or composition includes simultaneous administration and / or administration at different times. Concurrent administration also encompasses administration as a co-formulation or as separate compositions, including using different dosing frequencies or intervals, and the same or different routes of administration.

[0028] The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody." The term "antibody" is used herein in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, antibody fragments, so long as they exhibit biological activity, and antibody derivatives.

[0029] An "isolated" molecule or cell is a molecule or cell that has been identified and separated from at least one contaminating molecule or cell that is normally associated with the environment in which it was produced. Preferably, an isolated molecule or cell is not associated with all components associated with the environment in which it was produced. An isolated molecule or cell is in a form other than the form or context in which it is found in nature. Thus, an isolated molecule is distinct from a molecule that naturally occurs in a cell; an isolated cell is distinct from a cell that naturally occurs in a tissue, organ, or individual. In some embodiments, the isolated molecule is an anti-C1s, anti-C1q, or anti-C1r antibody of the present disclosure. In other embodiments, the isolated cell is a host cell or hybridoma cell that produces an anti-C1s, anti-C1q, or anti-C1r antibody of the present disclosure.

[0030] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free of association with all other contaminating components from its production environment. Contaminating components from its production environment, such as those resulting from recombinant transfected cells, are substances that would typically interfere with research, diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody, e.g., as determined by the Lowry method; (2) to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue, or preferably silver stain. An isolated antibody includes an antibody in situ in recombinant T cells, since at least one component of the antibody's natural environment is absent. Ordinarily, however, an isolated polypeptide or antibody will be prepared by a process that includes at least one purification step.

[0031] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The heavy and light chain variable domains are respectively referred to as "V H " and "V L " These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding sites.

[0032] The term "variable" refers to the fact that certain segments of the variable domains vary widely in sequence from one antibody to another. The V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration connected by three HVRs that form loops that connect and, in some cases, form part of the beta-sheet structure. The HVRs within each chain are held in close proximity to each other by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0033] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977), Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996) (the definitions include overlapping or subsets of amino acid residues when compared against each other). Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein.

[0034] As used herein, the terms "CDR-L1", "CDR-L2", and "CDR-L3" refer to the first, second, and third CDRs, respectively, in a light chain variable region. As used herein, the terms "CDR-H1", "CDR-H2", and "CDR-H3" refer to the first, second, and third CDRs, respectively, in a heavy chain variable region. As used herein, the terms "CDR-1", "CDR-2", and "CDR-3" refer to the first, second, and third CDRs, respectively, in the variable region of either chain.

[0035] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are typically synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained as a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can be produced using, for example, hybridoma methods (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., Nature, 352:624-628 (1991)), and the like. al., J.Mol.Biol.222:581-597(1992), Sidhu et al.,J.Mol.Biol.338(2):299-310(2004), Lee et al.,J.Mol.Biol.340(5):1073-1093(2004), Fellouse,Proc.Nat'l Acad.Sci.USA 101(34):12467-472 (2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741, Jakobovits et al. Proc. Nat'l Acad. Sci. USA 90:2551 (1993), Jakobovits et al., Nature 362:255-258 (1993), Bruggemann et al. Year in Immunol. 7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859 (1994), Morrison, Nature 368:812-813 (1994), Fishwild et al. Nature Biotechnol. 14:845-851 (1996), Neuberger, Nature Biotechnol. 14:826 (1996), and Lonberg and These can be produced by a variety of techniques, including the use of immunoglobulins such as IgG1, IgG2, and IgG3. (see Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0036] A "full-length antibody" is usually a heterotetrameric glycoprotein of about 150,000 daltons comprising two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H) followed by several constant domains. Each light chain has a variable domain (V L ) at its other end and a constant domain, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the variable domain of the light chain aligned with the variable domain of the heavy chain. It is believed that certain amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. The terms "full length antibody", "intact antibody" and "complete antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antibody fragment or antibody derivative. In particular, complete antibodies include those having heavy and light chains including an Fc region. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0037] An "antibody fragment" or "antigen-binding fragment" or "functional fragment" of an antibody comprises a portion of an intact antibody, preferably the F region of an antibody that retains or has the antigen-binding and / or variable region of the intact antibody or modified FcR binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; and linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)). Additional examples of antibody fragments include antibody derivatives, such as single-chain antibody molecules formed from antibody fragments, monovalent antibodies and multispecific antibodies.

[0038] An "antibody derivative" is any construct that contains the antigen-binding region of an antibody. Examples of antibody derivatives include single-chain antibody molecules formed from antibody fragments, monovalent antibodies, and multispecific antibodies.

[0039] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and one residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment contains the entire L chain plus the variable region domain of the H chain (VH ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to the two disulfide-linked Fab fragments with different antigen-binding activities and are still capable of cross-linking antigen. The Fab' fragment is H F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0040] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0041] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, and includes native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined as extending from the amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Thus, an intact antibody composition may include an antibody population with all K447 residues removed, an antibody population without the K447 residue removed, and an antibody population with a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies of the present disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0042] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0043] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, and preferably about 1 to about 5 amino acid substitutions. A variant Fc region herein preferably retains at least about 80% homology with a native sequence Fc region and / or the Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

[0044] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Further, a preferred FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors, where FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif ("ITIM") in its cytoplasmic domain. (See, e.g., M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. FcRs may increase the serum half-life of an antibody.

[0045] The in vivo binding to FcRn and serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having variant Fc regions are administered. WO2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, for example, Shields et al., J.Biol.Chem.9(2):6591-6604(2001).

[0046] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H chain and L chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0047] A "single-chain Fv", also abbreviated to "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains joined in a single polypeptide chain. Preferably, the sFv polypeptide comprises H and V L It further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0048] The term "diabody" refers to a diabody that is a peptide that binds V domains together to achieve inter-chain but not intra-chain V domain pairing, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H Domain and V L Refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (about 5-10 residues) between the domains. Bispecific diabodies are small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (about 5-10 residues) between the domains. H and V LDiabodies are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097, WO 1993 / 011161, WO / 2009 / 121948, WO / 2014 / 191493, Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48 (1993).

[0049] As used herein, "chimeric antibody" refers to antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived from an antibody generated, for example, by immunizing macaque monkeys with the antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies."

[0050] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, but the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs typically is no more than six in the H chain and no more than three in the L chain. The humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0051] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. The methods described in Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomice, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also Li et al., Proc. Nat'l. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B-cell hybridoma technology.

[0052] As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, an antibody contains six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the highest diversity among these six HVRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0053] Several HVR delineations are used and encompassed herein. The Kabat Complementarity Determining Regions (CDRs), HVRs, are based on sequence variability and are the most commonly used (Kabat et al., supra). Chothia, instead, refer to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are described below. [Table 1]

[0054] HVRs may include "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (preferred embodiment) (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these extended HVR definitions.

[0055] "Framework" or "FR" residues are those variable domain residues other than HVR residues as herein defined.

[0056] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by matching the sequence of the antibody with the "standard" Kabat numbered sequence at the regions of homology.

[0057] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system (see, e.g., U.S. Patent Publication No. 2010-280227).

[0058] An "amino acid modification" at a specified position refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent" to a specified residue means an insertion within 1-2 residues thereof. The insertion may be on the N-terminal or C-terminal side of the specified residue. A preferred amino acid modification herein is a substitution.

[0059] An "affinity matured" antibody is one that has one or more changes in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen compared to a parent antibody that does not have those change(s). In some embodiments, an affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are generated by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues has been described, for example, in Barbas et al. Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994), Schier et al. Gene 169:147-155 (1995), Yelton et al. J. Immunol. 155:1994-2004 (1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995), and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0060] As used herein, the term "specifically recognize" or "specifically bind" refers to a measurable and reproducible interaction, such as attraction or binding, between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically or preferentially binds to a target or epitope is an antibody that binds to this target or epitope with higher affinity, avidity, more readily, and / or with longer duration than it binds to other targets or other epitopes of targets. For example, it is understood that an antibody (or moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. An antibody that specifically binds to a target has at least about 10 3 M -1 or 10 4 M-1 , sometimes about 10 5 M -1 or 10 6 M -1 , and in other cases, about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 ~10 9 M -1 , or about 10 10 M -1 ~10 11 M -1 or higher. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0061] "Identity" as used herein indicates that the amino acid residue at any particular position in the aligned sequences is the same between the sequences. "Similarity" as used herein indicates that the amino acid residue at any particular position in the aligned sequences is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids that may often be substituted for one another include, but are not limited to: - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains); - lysine, arginine and histidine (amino acids with basic side chains); -Aspartate and glutamate (amino acids with acidic side chains); - asparagine and glutamine (amino acids with amide side chains); and -Cysteine ​​and methionine (amino acids with sulfur-containing side chains)

[0062] Degrees of identity and similarity can be readily calculated (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

[0063] As used herein, an "interaction" between a complement protein and a second protein includes, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, bonds, covalent bonds, and ionic bonds. As used herein, an antibody "inhibits an interaction" between two proteins if the antibody disrupts, reduces, or completely eliminates the interaction between the two proteins. An antibody of the present disclosure, or a fragment thereof, "inhibits an interaction" between two proteins if the antibody or a fragment thereof binds to one of the two proteins.

[0064] A "blocking," "antagonist," "inhibitory," or "neutralizing" antibody is an antibody that inhibits or reduces one or more biological activities of the antigen to which it binds, e.g., interaction with one or more proteins. In some embodiments, a blocking, antagonist, inhibitory, or "neutralizing" antibody substantially or completely inhibits one or more biological activities or interactions of an antigen.

[0065] The term "inhibitor" refers to a compound that has the ability to inhibit the biological function of a target biomolecule, for example, an mRNA or a protein, whether by decreasing the activity or expression of the target biomolecule. The inhibitor can be an antibody, a peptide, a protein, a nucleic acid, a small molecule, a gene editing agent, a base editing agent, or an epigenetic editing agent. The nucleic acid can be an antisense oligonucleotide, an miRNA, an miRNA inhibitor, an mRNA, an aptamer, or an antisense nucleic acid. The term "antagonist" refers to a compound that binds to a receptor and blocks or attenuates the biological response of the receptor. The term "inhibitor" can also refer to "antagonist".

[0066] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.

[0067] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances (e.g., an antibody and an antigen) and is expressed as a dissociation constant (KD). The affinity can be at least 1-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, or at least 1,000-fold higher, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of an antibody for a target protein can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more substances to dissociate upon dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0068] The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. For example, a subject anti-C1s antibody specifically binds to an epitope within the complement C1s protein. "Specific binding" refers to binding to an epitope that is at least about 10 -7 M or more, e.g., 5×10 -7 M, 10 -8 M, 5×10 -8 "Non-specific binding" refers to binding with an affinity of about 10 M or greater. -7 Affinity of less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity of M or similar.

[0069] The term "k" on" as used herein is intended to refer to the rate constant for association of an antibody to an antigen.

[0070] The term "k" off " as used herein, is intended to refer to the rate constant for dissociation of an antibody from the antibody / antigen complex.

[0071] The term “K D " as used herein, is intended to refer to the equilibrium dissociation constant of an antibody-antigen interaction.

[0072] As used herein, with respect to peptide, polypeptide, or antibody sequences, "percent (%) amino acid sequence identity" and "homology" refer to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art that are necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0073] A "biological sample" encompasses a variety of sample types obtained from an individual and may be used in diagnostic or monitoring assays. The definition includes blood and other liquid samples of biological origin, solid tissue samples, such as biopsies or tissue cultures or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in any way after their procurement, for example, by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses clinical samples, and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions, such as plasma and serum, and the like. The term "biological sample" also includes solid tissue samples, tissue culture samples, and cell samples.

[0074] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule that is normally associated with the environment in which it is produced. Preferably, an isolated nucleic acid is free of association with all components associated with the environment in which it is produced. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form other than the form or setting in which they are found in nature. Thus, isolated nucleic acid molecules are distinct from any nucleic acid encoding the polypeptides and antibodies herein that naturally occur in a cell.

[0075] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors," or simply "expression vectors." Typically, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector.

[0076] "Polynucleotide", or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be separated by non-nucleotide components. A polynucleotide can include modification(s) that are generated post-synthetically, such as conjugation to a label. Other types of modifications include, for example, "caps" which replace one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant sites such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to generate additional bonds to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH may be phosphorylated or replaced with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides may contain analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl, optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0077] As used herein, "carrier" includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Often, the physiologically acceptable carrier is an aqueous pH buffer. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0078] "Gene editing agents" as used herein are defined as gene editing agents, representative examples of which include CRISPR-associated nucleases such as Cas9 and Cpfl gRNA, the Argonaute family of endonucleases, clustered regularly interspaced short palindromic repeats (CRISPR) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endonucleases and / or exonucleases. See Schiffer, 2012, J Virol 88(17):8920-8936 (herein incorporated by reference).

[0079] As used herein, an "RNA interfering agent" is defined as any agent that disrupts or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, RNA molecules or fragments thereof that are homologous to the target biomarker gene of the present invention, small interfering RNAs (siRNAs), and nucleic acid molecules that include small molecules that prevent or inhibit the expression of a target biomarker nucleic acid by RNA interference (RNAi).

[0080] "RNA interference (RNAi)" is an evolutionarily conserved process in which expression or introduction of an RNA of identical or highly similar sequence to a target biomarker nucleic acid results in sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225) of messenger RNA (mRNA) transcribed from its target gene, thereby inhibiting expression of the target biomarker nucleic acid. In one embodiment, the RNA is double-stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes the processive cleavage of long dsRNA into double-stranded fragments called siRNAs. The siRNAs are incorporated into protein complexes that recognize and cleave the target mRNA. RNAi can also be initiated by introducing a nucleic acid molecule, such as a synthetic siRNA, shRNA, or other RNA interference agent, to inhibit or silence the expression of a target biomarker nucleic acid. As used herein, "inhibition of target biomarker nucleic acid expression" or "inhibition of marker gene expression" includes a reduction in either the expression or protein activity or level of a target biomarker nucleic acid or protein encoded by a target biomarker nucleic acid. The reduction can be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the expression of the target biomarker nucleic acid or the activity or level of a protein encoded by a target biomarker nucleic acid that is not targeted by an RNA interference agent.

[0081] In addition to RNAi, genome editing can be used to regulate the copy number or gene sequence of a biomarker of interest, such as constitutive or induced knockout or mutation of a biomarker of interest, such as complement pathway components such as C1q, C1r, and / or C1s. For example, CRISPR-Cas systems can be used for precise editing of genomic nucleic acids (e.g., to create non-functional or null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes can be expressed. For example, a vector containing only guide RNA can be administered to a Cas9 enzyme transgenic animal or cell. Similar strategies can be used (e.g., designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases). Such systems are well known in the art (see, e.g., U.S. Pat. No. 8,697,359; Sander and Joung (2014) Nat. Biotech. 32:347-355; Hale et al. (2009) Cell 139:945-956; Karginov and Hannon (2010) Mol. Cell 37:7; U.S. Patent Publication Nos. 2014 / 0087426 and 2012 / 0178169; Boch et al. (2011) Nat. Biotech. 29:135-136; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Weber et al. (2011) PLoS One (See, for example, Li et al. (2011) Nucl.Acids Res. 39:6315-6325, Zhang et al. (2011) Nat.Biotech. 29:149-153, Miller et al. (2011) Nat.Biotech. 29:143-148, Lin et al. (2014) Nucl.Acids Res. 42:e47.) Such genetic strategies can use constitutive or inducible expression systems, according to methods well known in the art.

[0082] "Piwi-interacting RNAs (piRNAs)" are the largest class of small non-coding RNA molecules. piRNAs form RNA-protein complexes through interactions with piwi proteins. These piRNA complexes have been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germline cells, particularly during spermatogenesis. They differ from microRNAs (miRNAs) in their size (26-31 nt instead of 21-24 nt), lack of sequence conservation, and increased complexity. However, like other small RNAs, piRNAs are thought to be involved in gene silencing, specifically silencing of transposons. The majority of piRNAs are antisense to transposon sequences, suggesting that transposons are piRNA targets. In mammals, the activity of piRNAs in transposon silencing appears to be most important during embryonic development, and in both C. elegans and humans, piRNAs are required for spermatogenesis. piRNAs have a role in RNA silencing through the formation of RNA-induced silencing complexes (RISCs).

[0083] An "aptamer" is an oligonucleotide or peptide molecule that binds to a specific target molecule. A "nucleic acid aptamer" is a nucleic acid species that is engineered through repeated rounds of in vitro selection, or equivalently, through SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. A "peptide aptamer" is an artificial protein that is selected or engineered to bind to a specific target molecule. These proteins consist of one or more peptide loops of variable sequence exhibited by a protein scaffold. They are typically isolated from combinatorial libraries and are often subsequently improved by rounds of directed mutation or variable region mutagenesis and selection. An evolution of peptide aptamers, "affimer proteins," are highly stable small proteins engineered to exhibit peptide loops that provide a high affinity binding surface for a specific target protein. They are low molecular weight 12-14 kDa proteins from the cysteine ​​protease inhibitor family of cystatins. Aptamers are useful in biotechnology and therapeutic applications, offering molecular recognition properties comparable to antibodies, a commonly used biomolecule. In addition to their discriminatory recognition, aptamers offer advantages over antibodies because they can be fully engineered in vitro, are easily produced by chemical synthesis, have desirable storage properties, and induce little or no immunogenicity in therapeutic applications.

[0084] "Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA", is defined as an agent that functions to inhibit expression of a target biomarker nucleic acid, for example, by RNAi. siRNA may be chemically synthesized, produced by in vitro transcription, or produced within a host cell. In one embodiment, the siRNA is a double-stranded RNA (dsRNA) molecule about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides in length, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may contain 3' and / or 5' overhangs on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhangs is independent between the two strands, i.e., the length of the overhang on one strand is independent of the length of the overhang on the second strand. Preferably, the siRNA is capable of promoting RNA interference through degradation of the target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

[0085] The term "preventing" is art-recognized and, when used in relation to conditions such as muscular dystrophy, is well understood in the art and includes administration of a composition to reduce the frequency or severity of, or delay the onset of, one or more symptoms of a medical condition in a subject compared to a subject not administered the composition. Thus, preventing muscular dystrophy includes, for example, preserving muscle strength in a population of treated patients compared to a control population not treated, for example, by a statistically and / or clinically significant amount. Similarly, preventing muscular dystrophy includes reducing the likelihood that a treated patient will develop muscular dystrophy or an associated condition relative to a non-treated patient.

[0086] The term "slowing or blocking progression" as used herein refers to slowing or stopping the progression of a condition such as muscular dystrophy. Disease progression is assessed by describing the natural history of the disease and measuring and monitoring functional outcomes over a period of time. For example, as the disease progresses, muscle weakness and wasting (atrophy) progresses. Administration of the compositions described herein can slow or block the progression of muscle weakness and wasting.

[0087] The term "subject" as used herein refers to a living mammal and may be used interchangeably with the term "patient". Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees, and other ape and monkey species; farm animals, such as cows, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, and the like. The term does not denote a particular age or sex.

[0088] As used herein, the term "treating" or "treatment" includes reducing, arresting, or reversing the symptoms, clinical signs, or underlying pathology of a condition, stabilizing or ameliorating the subject's condition or making it less likely that the subject's condition will worsen to the same extent as if the subject had not received treatment.

[0089] The term "therapeutically effective amount" of a compound in the context of a subject treatment method refers to the amount of compound(s) in a preparation that, when administered as part of a desired dosing regimen (for a mammal, preferably a human), relieves symptoms, ameliorates pathology, or delays the onset of a disease or condition, for example, in accordance with clinically acceptable standards for treating a disease or condition or for cosmetic purposes, with a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutically effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, as well as the ability of the antibody to elicit a desired response in an individual.

[0090] As used herein, an individual who is "at risk" of developing a particular disease, disorder, or condition may or may not show detectable disease or disease symptoms, and may or may not show detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" means that an individual has one or more risk factors, which are measurable parameters that correlate with the development of a particular disease, disorder, or condition, as known in the art. Individuals who have one or more of these risk factors are more likely to develop a particular disease, disorder, or condition than individuals who do not have one or more of these risk factors.

[0091] "Chronic" administration refers to the administration of a pharmaceutical agent(s) continuously, as opposed to in an acute manner, so as to maintain an initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is cyclic / periodic in nature, rather than administered continuously without interruption.

[0092] As used herein, administration "in conjunction with" another compound or composition includes simultaneous administration and / or administration at different times. Concurrent administration also encompasses administration as a co-formulation or as separate compositions, including using different dosing frequencies or intervals, and the same or different routes of administration.

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety, including, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMA Usubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames and G.R. Taylor eds. (1995)); Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press.Wiley and Sons, Handbook of Experimental Immunology (DMWeir and CCBlackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JEColigan et al. al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., J.B. Lippincott Company, 1993), are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which these publications are cited.

[0094] Classical complement inhibitors The present disclosure broadly relates to methods of preventing, reducing the risk of developing, slowing or blocking the progression of, or treating Duchenne muscular dystrophy ("DMD"), Becker muscular dystrophy ("BMD"), limb-girdle muscular dystrophies (LGMD) (including sarcoglycanopathies, dystroglycanopathies, and dysferlinopathies), collagen VI-related disorders (including Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD)), congenital muscular dystrophies (CMDs) and congenital myopathies, and distal muscular dystrophies / myopathies (including Miyoshi myopathy). The method includes administering to the subject an inhibitor of the classical complement pathway, such as a C1 complex inhibitor, a C1q inhibitor, a C1s inhibitor, a C1r inhibitor, or a C1 complex inhibitor. The inhibitor can be an antibody, a peptide, a protein, a nucleic acid, a small molecule, a gene editing agent, a base editing agent, or an epigenetic editing agent. The nucleic acid can be an antisense oligonucleotide, miRNA, miRNA inhibitor, mRNA, aptamer, or antisense nucleic acid. An inhibitor can refer to a compound that has the ability to inhibit the biological function of a target biomolecule, whether by reducing the activity or expression of the target biomolecule.

[0095] Inhibitors can block activation of the complement cascade, block expression of specific complement proteins, interfere with signaling molecules that induce complement activation, upregulate expression of complement inhibitors, and otherwise interfere with the role of complement.

[0096] Based on the molecular structure of the C1 complex, C1q, C1r, or C1s, or the variable region of an anti-C1q, C1r, or C1s antibody, molecular modeling and rational molecular design can be used to generate and screen small molecules that mimic the molecular structure of the binding region of the antibody and / or inhibit the activity of C1 complex, C1q, C1r, or C1s. These small molecules can be peptides, peptidomimetics, oligonucleotides, or organic compounds. The mimetic molecules can be used as inhibitors of complement activation. Alternatively, large-scale screening procedures commonly used in the field can be used to isolate suitable small molecules from libraries of combinatorial compounds.

[0097] Some molecules that inhibit the activity of complement are known. In addition to known compounds, suitable inhibitors can be screened by the methods described herein. As mentioned above, normal cells can produce proteins that block complement activity, such as CD59, C1 inhibitor, etc. In some embodiments of the present disclosure, complement is inhibited by upregulating the expression of genes that code for such polypeptides.

[0098] Modifications of molecules that block complement activation are also known in the art. For example, such molecules include, but are not limited to, modified complement receptors such as soluble CR1. The mature protein of the most common allotype of CR1 contains 1998 amino acid residues: an extracellular domain of 1930 residues, a transmembrane region of 25 residues, and a cytoplasmic domain of 43 residues. The entire extracellular domain is composed of 30 repeating units, termed short consensus repeats (SCRs) or complement control protein repeats (CCPRs), each consisting of 60-70 amino acid residues. Recent data indicate that C1q binds specifically to human CR1. Thus, CR1 recognizes all three complement opsonins, namely C3b, C4b, and C1q. Soluble versions of recombinant human CR1 (sCR1), lacking the transmembrane and cytoplasmic domains, have been produced and shown to retain all known functions of native CR1. Several types of human C1q receptors (C1qR) have been described. These include a ubiquitously distributed 60-67 kDa receptor termed cC1qR because it binds to the collagen-like domain of C1q. This C1qR variant was shown to be calreticulin, a 126 kDa receptor that regulates monocyte phagocytosis. gC1qR is not a membrane-bound molecule but is a secreted soluble protein with affinity for the globular domain of C1q and may function as a fluid-phase regulator of complement activation.

[0099] Decay-accelerating factor (DAF) (CD55) is composed of four SCRs and a serine / threonine-rich domain capable of extensive O-linked glycosylation. DAF is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor and acts by dissociating the C3 and C5 convertases through its ability to bind C4b and C3b. A soluble version of DAF (sDAF) has been shown to inhibit complement activation.

[0100] C1 inhibitor, a member of the "serpin" family of serine protease inhibitors, is a heavily glycosylated plasma protein that prevents fluid-phase C1 activation. C1 inhibitor regulates the classical pathway of complement activation by blocking the active sites of C1r and C1s, dissociating them from C1q.

[0101] Peptide inhibitors of complement activation include C5a, and other inhibitory molecules include fucans.

[0102] All sequences recited in this disclosure are incorporated by reference from U.S. Patent No. 10,316,081, U.S. Patent Application No. 14 / 890,811, U.S. Patent No. 8,877,197, U.S. Patent No. 9,708,394, U.S. Patent No. 10,723,788, U.S. Patent No. 9,562,106, U.S. Patent No. 10,450,382, U.S. Patent No. 10,457,745, International Patent Application No. PCT / US2018 / 022462, each of which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0103] Anti-C1 complex antibody In other embodiments, the inhibitor of the classical complement pathway may be an anti-C1 complex antibody, optionally inhibiting C1r or C1s activation or preventing their ability to act on C2 or C4, e.g., the anti-C1 complex antibody binds to a combination epitope in the C1 complex, the combination epitope including both C1q and C1s, both C1q and C1r, both C1r and C1s, or amino acids of each of C1q, C1r, and C1s. The antibody may be a monoclonal antibody. In some embodiments, the antibody inhibits cleavage of C4 and does not inhibit cleavage of C2, or inhibits cleavage of C2 and does not inhibit cleavage of C4.

[0104] In some embodiments, the antibody binds to mammalian C1q, C1r, or C1s, or binds to human C1q, C1r, or C1s, hi some embodiments, the antibody binds to a mammalian C1 complex.

[0105] Anti-complement C1q antibodies The anti-C1q antibodies disclosed herein are potent inhibitors of C1q and can be dosed for continuous inhibition of C1q function for any period of time and then optionally discontinued to allow restoration of normal C1q function at a time when its activity may be important. The results obtained with the anti-C1q antibodies disclosed herein in animal studies can be readily advanced to the clinic using humanized or human antibodies, as well as fragments and / or derivatives thereof.

[0106] C1q is a large multimeric protein of 460 kDa consisting of 18 polypeptide chains (6 C1q A chains, 6 C1q B chains, and 6 C1q C chains). The C1r and C1s complement proteins bind to the C1q tail region to form the C1 complex (C1qr2s2).

[0107] The anti-C1q antibodies of the present disclosure specifically recognize complement factor C1q and / or C1q in the C1 complex of the classical complement activation pathway. The bound complement factor can be from any organism that has a complement system, including, but not limited to, any mammalian organism, such as human, mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig.

[0108] As used herein, a "C1 complex" refers to a protein complex that may include, but is not limited to, one C1q protein, two C1r proteins, and two C1s proteins (e.g., C1qr 2 s 2 )

[0109] The anti-C1q antibodies disclosed herein can inhibit C1 complex formation.

[0110] As used herein, "complement factor C1q" refers to both the wild-type sequence and the naturally occurring variant sequence.

[0111] A non-limiting example of a complement factor C1q recognized by the antibodies of the present disclosure is human C1q, which comprises three polypeptide chains A, B, and C: C1q, Chain A (Homo sapiens), Accession No. Protein Database: NP_057075.1; GenBank number: NM_015991: >gi|7705753|ref|NP_057075.1|Complement C1q Subcomponent Subunit A Precursor [Homo sapiens] (SEQ ID NO:1) MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRN PPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA.

[0112] C1q, Chain B (Homo sapiens), Accession No. Protein Database: NP_000482.3; GenBank No.: NM_000491.3: >gi|87298828|ref|NP_000482.3|Complement Clq Subcomponent Subunit B Precursor [Homo sapiens] (SEQ ID NO:2) MMMKIPWGSIPVLMLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSAT RTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA.

[0113] C1q, Chain C (Homo sapiens), Accession No. Protein Database: NP_001107573.1; GenBank number: NM_001114101.1: >gi|166235903|ref|NP_001107573.1|Complement C1q Subcomponent Subunit C Precursor [Homo sapiens] (SEQ ID NO:3) MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFT VTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD.

[0114] Thus, an anti-C1q antibody of the disclosure may bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of a C1q protein. In some embodiments, an anti-C1q antibody of the disclosure binds to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of human C1q or a homolog thereof, e.g., mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig C1q. In some embodiments, the anti-C1q antibody is a human antibody, a humanized antibody, a chimeric antibody, or a fragment or derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment.

[0115] Light and heavy chain variable domain sequences of antibody M1 All sequences referenced in connection with antibody M1 are incorporated by reference from US Pat. No. 9,708,394, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0116] The nucleic acid and amino acid sequences encoding the light chain variable and heavy chain variable domains of antibody M1 were determined using standard techniques. The amino acid sequence of the light chain variable domain of antibody M1 is as follows:

[0117] TIFF2024539141000002.tif25170

[0118] The hypervariable regions (HVRs) of the light chain variable domains are shown in bold and underlined text. In some embodiments, HVR-L1 of the M1 light chain variable domain has the sequence RASKSINKYLA (SEQ ID NO:5), HVR-L2 of the M1 light chain variable domain has the sequence SGSTLQS (SEQ ID NO:6), and HVR-L3 of the M1 light chain variable domain has the sequence QQHNEYPLT (SEQ ID NO:7).

[0119] The amino acid sequence of the heavy chain variable domain of antibody M1 is:

[0120] TIFF2024539141000003.tif31170

[0121] The hypervariable regions (HVRs) of the heavy chain variable domains are shown in bold and underlined text. In some embodiments, HVR-H1 of the M1 heavy chain variable domain has the sequence GYHFTSYWMH (SEQ ID NO:9), HVR-H2 of the M1 heavy chain variable domain has the sequence VIHPNSGSINYNEKFES (SEQ ID NO:10), and HVR-H3 of the M1 heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQ ID NO:11).

[0122] The nucleic acid sequence encoding the light chain variable domain was determined to be: GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAA TCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAACATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 12).

[0123] The nucleic acid sequence encoding the heavy chain variable domain was determined to be: CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACCATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGTGATTCATCCTAATAGTGGTAGTATTAACTACAATGAG AAGTTCGAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCGGCGGTCTATTATTGTGCAGGAGAGAGAGATTCTACGGAGGTTCTCCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 13).

[0124] Deposit of materials The following materials have been deposited in accordance with the Budapest Treaty with the American Type Culture Collection, ATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC): [Table 2]

[0125] A hybridoma cell line (mouse hybridoma C1qM1 7788-1(M)051613) producing the M1 antibody has been deposited with the ATCC under conditions that ensure that access to the culture will be available during the pendency of the patent application and for a period of 30 years, or 5 years after the latest claim, or until the life of the patent, whichever is longer. The deposit will be replaced if it becomes non-viable during that period. The deposit will be available as required by foreign patent laws in countries in which counterpart applications of this application, or progeny thereof, are filed. It should be understood, however, that the availability of the deposit does not constitute a license to practice the invention in derogation from patent rights granted by governmental action.

[0126] In some embodiments, the amino acid sequences of the light chain variable domain and the heavy chain variable domain comprise one or more of SEQ ID NO:5 for HVR-L1, SEQ ID NO:6 for HVR-L2, SEQ ID NO:7 for HVR-L3, SEQ ID NO:9 for HVR-H1, SEQ ID NO:10 for HVR-H2, and SEQ ID NO:11 for HVR-H3.

[0127] The antibody may comprise a light chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO:4, preferably retaining HVR-L1 RASKSINKYLA (SEQ ID NO:5), HVR-L2 SGSTLQS (SEQ ID NO:6), and HVR-L3 QQHNEYPLT (SEQ ID NO:7). The antibody may comprise a heavy chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO:8, preferably retaining HVR-H1 GYHFTSYWMH (SEQ ID NO:9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO:10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO:11).

[0128] Humanized anti-complement C1q antibody The humanized antibodies of the present disclosure specifically bind to complement factor C1q and / or C1q protein in the C1 complex of the classical complement pathway. The humanized anti-C1q antibodies may specifically bind to human C1q, human and mouse C1q, rat C1q, or human C1q, mouse C1q, and rat C1q.

[0129] All sequences referenced in relation to the humanized anti-C1q antibodies are incorporated by reference from US Pat. No. 10,316,081, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0130] In some embodiments, the human heavy chain constant region is a human IgG4 heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% homology to SEQ ID NO: 47. The human IgG4 heavy chain constant region may comprise an Fc region having one or more modifications and / or amino acid substitutions according to Kabat numbering. In such cases, the Fc region comprises an amino acid substitution of leucine to glutamate at position 248, such a substitution inhibits the Fc region from interacting with an Fc receptor. In some embodiments, the Fc region comprises an amino acid substitution of serine to proline at position 241, such a substitution prevents arm switching in the antibody.

[0131] The amino acid sequence of the human IgG4 (S241P L248E) heavy chain constant domain is: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47).

[0132] The antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 31-34, or an amino acid sequence having at least about 90% homology to an amino acid sequence selected from any one of SEQ ID NOs: 31-34. In certain such embodiments, the light chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 35-38, or an amino acid sequence having at least about 90% homology to an amino acid sequence selected from any one of SEQ ID NOs: 35-38.

[0133] The amino acid sequence of the heavy chain variable domain variant 1 (VH1) is:

[0134] TIFF2024539141000005.tif31170The hypervariable regions (HVRs) of VH1 are shown in bold and underlined text.

[0135] The amino acid sequence of the heavy chain variable domain variant 2 (VH2) is:

[0136] TIFF2024539141000006.tif31170 Hypervariable regions (HVRs) of VH2 are shown in bold and underlined text.

[0137] The amino acid sequence of the heavy chain variable domain variant 3 (VH3) is:

[0138] TIFF2024539141000007.tif31170 The hypervariable regions (HVRs) of VH3 are shown in bold and underlined text.

[0139] The amino acid sequence of the heavy chain variable domain variant 4 (VH4) is:

[0140] TIFF2024539141000008.tif31170 The hypervariable regions (HVRs) of VH4 are shown in bold and underlined text.

[0141] The amino acid sequence of the kappa light chain variable domain variant 1 (Vκ1) is:

[0142] TIFF2024539141000009.tif25170The hypervariable regions (HVRs) of Vκ1 are shown in bold and underlined text.

[0143] The amino acid sequence of the kappa light chain variable domain variant 2 (Vκ2) is:

[0144] TIFF2024539141000010.tif25170The hypervariable regions (HVRs) of Vκ2 are shown in bold and underlined text.

[0145] The amino acid sequence of the kappa light chain variable domain variant 3 (Vκ3) is:

[0146] TIFF2024539141000011.tif25170The hypervariable regions (HVRs) of Vκ3 are shown in bold and underlined text.

[0147] The amino acid sequence of the kappa light chain variable domain variant 4 (Vκ4) is:

[0148] TIFF2024539141000012.tif25170The hypervariable regions (HVRs) of Vκ4 are shown in bold and underlined text.

[0149] The antibody may comprise a light chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NOs: 35-38, while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6), and HVR-L3 QQHNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NOs: 31-34, while retaining preferably HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0150] In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 35 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 36 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 32. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 37 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 38 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 34.

[0151] In some embodiments, the humanized anti-C1q antibody of the present disclosure comprises a heavy chain variable region containing a Fab region and a heavy chain constant region containing an Fc region, wherein the Fab region specifically binds to a C1q protein of the present disclosure, while the Fc region is incapable of binding to a C1q protein. In some embodiments, the Fc region is derived from a human IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc region is incapable of inducing complement activity and / or incapable of inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc region comprises one or more modifications, including, but not limited to, an amino acid substitution. In certain embodiments, the Fc region of the humanized anti-C1q antibody of the present disclosure comprises an amino acid substitution at position 248 according to the Kabat numbering convention or a position corresponding to position 248 according to the Kabat numbering convention, and / or at position 241 according to the Kabat numbering convention or a position corresponding to position 241 according to the Kabat numbering convention. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 inhibits the Fc region from interacting with an Fc receptor. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 is a leucine to glutamate amino acid substitution. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 prevents an arm switch in the antibody. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 is a serine to proline amino acid substitution. In certain embodiments, the Fc region of a humanized anti-C1q antibody of the present disclosure comprises an amino acid sequence of SEQ ID NO:47, or an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homology to the amino acid sequence of SEQ ID NO:47.

[0152] Anti-C1q Fab fragment Before the advent of recombinant DNA technology, proteolytic enzymes (proteases) that cleave the polypeptide sequence were used to break down the structure of antibody molecules and determine which parts of the molecule are responsible for their various functions. Limited digestion with the protease papain cleaves an antibody molecule into three fragments. Two of the fragments, known as Fab fragments, are identical and contain antigen-binding activity. The Fab fragments correspond to two identical arms of the antibody molecule, each of which contains the V of the heavy chain. H and C H The Fab fragment consists of a complete light chain paired with an Fc domain. The other fragment was initially observed to contain no antigen-binding activity but was easily crystallized, and for this reason was named the Fc fragment (fragment crystallizable). When comparing Fab molecules with IgG molecules, Fabs were found to be superior to IgG for certain in vivo applications due to their higher mobility and tissue penetration capabilities, their reduced circulating half-life, their ability to bind antigen monovalently without mediating antibody effector functions, and their lower immunogenicity.

[0153] Fab molecules contain the constant domain C H 2 and C H It is an artificial approximately 50 kDa fragment of an Ig molecule with the heavy chain shortened by 3. Two heterophils (V L -V H and C L -C H 1) Domain interactions underlie the structure of the two chains of the Fab molecule, which is L and C. H 1. Fab and IgG have six complementarity determining regions (CDRs), three each of which is V L and V H The CDRs define the hypervariable antigen-binding site of the antibody. The highest sequence variation is found in LCDR3 and HCDR3, which in the natural immune system are V and VD, respectively. L and J L Gene or V H , DH and J H They are generated by genetic rearrangement. LCDR3 and HCDR3 typically form the core of the antigen-binding site. The conserved regions connecting and presenting the six CDRs are called framework regions. In the three-dimensional structure of the variable domain, the framework regions form a sandwich of two opposing antiparallel β-sheets linked on the outside by the hypervariable CDR loops and on the inside by conserved disulfide bridges. This unique combination of stability and versatility of the antigen-binding sites of Fab and IgG underscores their success in clinical practice for the diagnosis, monitoring, prevention, and treatment of diseases.

[0154] All anti-C1q antibody Fab fragment sequences are incorporated by reference from US Pat. No. 10,723,788, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0155] In certain embodiments, the present disclosure provides a method for the production of a heavy chain (V H / C H 1) and light chain (V L / C L ), wherein the anti-C1q antibody Fab fragment binds to the C1q protein and comprises six complementarity determining regions (CDRs) (three of which are V L and V H The heavy chain of the antibody Fab fragment is truncated after the first heavy chain domain of IgG1 (SEQ ID NO: 39) and comprises the following amino acid sequence:

[0156] [Outside 11] TIFF2024539141000013.tif43170

[0157] The complementarity determining regions (CDRs) of SEQ ID NO:1 are shown in bold and underlined text.

[0158] The light chain domain of the antibody Fab fragment comprises the following amino acid sequence (SEQ ID NO:40):

[0159] [Outside 12] TIFF2024539141000014.tif43170

[0160] The complementarity determining regions (CDRs) of SEQ ID NO:2 are shown in bold and underlined text.

[0161] Anti-complement C1s antibody Suitable inhibitors include antibodies that bind to complement C1s protein (i.e., anti-complement C1s antibodies, also referred to herein as anti-C1s antibodies and C1s antibodies) and nucleic acid molecules encoding such antibodies. Complement C1s is an attractive target because it is upstream in the complement cascade and has a narrow range of substrate specificity. Furthermore, it is possible to obtain antibodies (such as, but not limited to, monoclonal antibodies) that specifically bind to the activated form of C1s.

[0162] Examples of anti-C1s antibodies are disclosed in US Patent Application Serial No. 14 / 890,811 and US Pat. No. 8,877,197, which are incorporated herein by reference for the antibodies and related compositions they disclose.

[0163] In certain aspects, disclosed herein is a method of administering an anti-C1s antibody. The antibody can be a murine antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3, and the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3 of the murine anti-human C1s monoclonal antibody 5A1 produced by the hybridoma cell line deposited with the ATCC on 5 / 15 / 2013 or its progeny (ATCC Accession No. PTA-120351). In other embodiments, the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3, and the heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3 of the mouse anti-human C1s monoclonal antibody 5C12 produced by the hybridoma cell line deposited with the ATCC on 5 / 15 / 2013 or its progeny (ATCC Accession No. PTA-120352).

[0164] Nucleic Acids, Vectors and Host Cells Antibodies suitable for use in the methods of the present disclosure may be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In some embodiments, an isolated nucleic acid having a nucleotide sequence encoding any of the antibodies of the present disclosure is provided. Such nucleic acids include those encoding the V domain of anti-C1q, anti-C1r, or anti-C1s antibodies. L / C L and / or V H / C H In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. Host cells containing such nucleic acids may also be provided. The host cells may encode an amino acid sequence containing (1) the V of an antibody. L / C L Amino acid sequence containing and V of antibody H / C H (2) a vector containing a nucleic acid encoding an amino acid sequence containing the V L / C L A first vector containing a nucleic acid encoding an amino acid sequence containing H / C H The host cell may contain (e.g., be transduced with) a second vector containing a nucleic acid encoding an amino acid sequence containing 1. In some embodiments, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In some embodiments, the host cell is a bacterium, such as E. coli.

[0165] Disclosed herein is a method for producing an anti-C1q, anti-C1r or anti-C1s antibody. The method includes culturing a host cell of the disclosure that contains a nucleic acid encoding an anti-C1q, anti-C1r or anti-C1s antibody under conditions suitable for expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or host cell medium).

[0166] Antibody Screening Candidate antibodies can be screened for their ability to modulate complement activation. Such screening can be performed using in vitro models, genetically altered cells or animals, or purified proteins. A variety of assays, such as in vitro culture systems, can be used for this purpose.

[0167] Candidate antibodies can also be identified using computer-based modeling, by binding assays, etc. Various in vitro models can be used to determine whether an antibody binds or otherwise affects complement activity. Such candidate antibodies can be tested by contacting them with plasma from healthy donors and determining complement activation (e.g., by antigen C3c capture ELISA).

[0168] Usually, multiple assay mixtures are run in parallel at different antibody concentrations to obtain differential responses to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., zero concentration or below the level of detection.

[0169] Pharmaceutical Compositions and Administration The complement inhibitors (eg, antibodies, antibody fragments and / or antibody derivatives) of the present disclosure can be administered in the form of a pharmaceutical composition.

[0170] Therapeutic formulations of the inhibitors (e.g., antibodies, antibody fragments and / or antibody derivatives) of the present disclosure may be prepared in the form of a lyophilized formulation or an aqueous solution for storage by mixing the inhibitors having the desired purity with any pharma- ceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.

[1980] ). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as Examples of suitable surfactants include serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0171] The inhibitors may also be incorporated into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0172] The formulations used for administration can be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0173] The antibodies, antibody fragments and / or antibody derivatives and compositions of the disclosure are typically administered by a variety of routes, including, but not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration. Parenteral routes of administration include intramuscular, intravenous, intraarterial, intraperitoneal, intrathecal, or subcutaneous administration.

[0174] The pharmaceutical composition may also include a pharma- ceutically acceptable non-toxic carrier of a diluent, defined as a vehicle commonly used to formulate a pharmaceutical composition for animal or human administration, depending on the desired formulation. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, and the like. The composition may also include additional substances for approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.

[0175] The compositions may also include any of a variety of stabilizing agents, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be complexed with a variety of well-known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance other pharmacokinetic and / or pharmacodynamic properties, or enhance solubility or uptake).

[0176] Toxicity and therapeutic efficacy of an active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred.

[0177] The pharmaceutical compositions described herein can be administered in a variety of different ways, examples include administering the composition containing a pharma- ceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.

[0178] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives.

[0179] The components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, usually at least analytical grade, more typically at least pharmaceutical grade). Furthermore, compositions intended for parenteral use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic substances, particularly any endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also typically substantially isotonic and produced under GMP conditions.

[0180] The effective amount of a therapeutic composition given to a particular patient may depend on a variety of factors, some of which may vary from patient to patient. A competent clinician can determine the effective amount of a therapeutic agent to administer to a patient. The dosage of the agent depends on the treatment, the route of administration, the nature of the therapeutic agent, the patient's sensitivity to the therapeutic agent, and the like. Using LD50 animal data and other information, a clinician can determine the maximum safe dose for an individual, depending on the route of administration. Using routine techniques, a competent clinician can optimize the dosage of a particular therapeutic composition during the course of routine clinical trials. The composition may be administered to a subject in a series of multiple doses. With respect to therapeutic compositions, periodic periodic administration may sometimes be required or desirable. Treatment regimens vary depending on the agent; for example, some agents may be taken on a once-daily or twice-daily basis for an extended period of time, while more selective agents may be administered to be taken once-daily, twice-daily, twice-weekly, once-weekly, etc., for a more defined period of time, such as, for example, 1, 2, 3 or more days, a week or more, a month or more, etc.

[0181] Treatment The present disclosure broadly relates to a method for preventing, reducing the risk of developing, slowing or blocking the progression of, or treating Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophies (LGMDs) (including sarcoglycanopathies, dystroglycanopathies, and dysferlinopathies), collagen VI-related disorders (including Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD)), congenital muscular dystrophies (CMDs) and congenital myopathies, and distal muscular dystrophies / myopathies (including Miyoshi myopathy). The method includes administering to a subject an inhibitor of the classical complement pathway, such as a C1 complex inhibitor, a C1q inhibitor, a C1s inhibitor, or a C1r inhibitor. The inhibitor can be an antibody, a peptide, a protein, a nucleic acid, a small molecule, a gene editing agent, a base editing agent, or an epigenetic editing agent. The nucleic acid may be an antisense oligonucleotide, miRNA, miRNA inhibitor, mRNA, aptamer, or antisense nucleic acid. Inhibitor may refer to a compound that has the ability to inhibit the biological function of a target biomolecule, whether by decreasing the activity or expression of the target biomolecule. Such a method includes administering a C1q inhibitor to a subject. Numerous embodiments are further provided that may be applied to any aspect of the invention described herein. For example, in some embodiments, the C1q inhibitor is an antibody, an aptamer, an antisense nucleic acid, or a gene editing agent. In some embodiments, the inhibitor is an anti-C1q antibody. The anti-C1q antibody may inhibit the interaction between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s, or may promote the clearance of C1q from the circulation or tissues.

[0182] It is contemplated that the compositions may be obtained and used under the guidance of a physician for in vivo use. The dosage of the therapeutic formulation may vary widely depending on the nature of the disease, the frequency of administration, the mode of administration, clearance of the drug from the host, and the like. EXAMPLES

[0183] Example 1: Materials and Methods Unless otherwise stated, the methods and materials described in this Example 1 were used in the examples described herein.

[0184] mouse Animals were maintained with free access to food and water and kept at a constant temperature (19-22°C) with a 12:12-h light / dark cycle. C57BL / 6J mice (Charles River Laboratories) were used as wild-type (WT) animals. mdmdx-4Cv / J Mice (Charles River Laboratories, hereafter referred to as mdx 4Cv For experiments using anti-C1q blocking antibody (mouse antibody), Pax7-CreER tm Male R26R YFP / YFP The female (Jackson Laboratory) was crossed with Pax7Cre ER / WT ;R26R YFP / WT These breeders were female mdx 4Cv / 4Cv To generate male experimental animals (Pax7Cre ER / WT ;R26R YFP / WT ;mdx 4Cv Tamoxifen (T5648 Sigma) was dissolved at 50 mg / ml in 92.5% corn oil / 7.5% ethanol, and 2.5 mg was administered intraperitoneally to 54-day-old experimental mice daily for 8 days. + / - ;C1qa FL / FL ;mdx 4Cv For generation, C1qa FL / FL (The Jackson Laboratory) Male C1qa WT / WT ;mdx 4Cv / 4Cv Mating to females, C1qa FL / WT ;mdx 4Cv These males were then used as C1qa WT / WT ;mdx 4Cv / 4Cv Mating to females, C1qa FL / WT ;mdx 4Cv / 4Cv Obtain a female and then C1qa FL / WT ;mdx 4Cv Mating with males, C1qa FL / FL ;mdx 4Cv / 4CvGot a female. Lyz2Cre + / + (The Jackson Laboratory) Male Lyz2Cre - / - ;mdx 4Cv / 4Cv Mate to a female and Lyz2Cre + / - ;mdx 4Cv These breeders were C1qa FL / FL ;mdx 4Cv / 4Cv Mate to a female and Lyz2Cre + / - ;C1qa FL / WT ;mdx 4Cv Finally, we obtained C1qa FL / FL ;mdx 4Cv / 4Cv Lyz2Cre the female + / - ;C1qa FL / WT ;mdx 4Cv The male experimental animals (Lyz2Cre + / - ;C1qa FL / FL ;mdx 4Cv , herein C1qa KO ;mdx 4Cv Mice were obtained from the mice (referred to as "mice"). Genotyping was performed using the primers listed in Table 1. [Table 3]

[0185] Behavioral testing Prior to behavioral testing, mice were subjected to regular handling by the same operator to limit stress levels. Tests were consistently performed at the same time during the mouse dark phase. All behavioral tests were performed blinded.

[0186] Limb hanging test The limb hanging test protocol was adapted from Treat-NDM Neuromuscular Network SOP DMD_M2.2.1.005. Prior to testing, mice were weighed to normalize by body weight. A 40 cm × 30 cm metal grid was placed 40 cm above the desk with sufficient bedding (i.e., paper and wood chips) to ensure a soft landing. Mice were placed on the grid and allowed to acclimate to this environment for 3–5 s, after which the grid was inverted and held at a height of 40 cm. If mice fell off the grid before the predefined time limit (900 s), they were immediately given two more trials. The total hanging time was recorded. Tests were repeated three times, every other day.

[0187] Double grip test The bilimb grip strength test protocol was adapted from Treat-NMD Neuromuscular Network SOP DMD_M2.2.001. Prior to testing, mice were weighed to normalize for body weight. Only the forelimbs of the mouse were attached to a metal trapezoid connected to a force transducer and electronic unit (47200-Grip Strength Meter-Ugo Basile). The mouse's tail was gently pulled. Maximum grip strength and total test time (i.e., total time the mouse was attached to the trapezoid) were recorded four times and the mean of these measurements was calculated. Tests were repeated six times (C1qaKO Cntr mice: three trials on three consecutive days, then three further trials on five alternating days) or three times (WT mice alternating).

[0188] Open field test The open field test protocol was adapted from Treat-NDM Neuromuscular Network SOP DMD_M2.2.1.002. Immediately after the limb hanging test, mice were placed in a 40 cm × 40 cm arena and allowed to move freely and explore for 5 or 6 min. The test was recorded and behavioral data were analyzed using ANY-maze software or EthoVision XT software. The test was repeated three times every other day.

[0189] Rotarod test The rotarod test was performed using a LE8205 Panlab Harvard Apparatus. Before the test, the mice were weighed to normalize by body weight. The speed of the rotarod was set at a constant value of 6 rpm. After 1 hour, the speed was increased to a constant value of 12 rpm. If the mice fell off the rod, they were immediately tested three more times. The total walking time was recorded. The test was repeated for 6 days (3 tests on 3 consecutive days, then 3 more tests on alternate days after 5 days).

[0190] Collection and preparation of plasma and serum Mice were heated under a lamp for a few minutes to increase blood flow. The area of ​​the submandibular vein was pierced with a needle tip and blood flow from the cheek was collected in a test tube. Blood samples were incubated at room temperature for 1 hour and centrifuged at 2.000g for 10 minutes at 4°C. The supernatant (serum) was collected. Alternatively, blood was collected in EDTA-treated tubes. Samples were centrifuged at 2.000g for 10 minutes at 4°C and the supernatant (plasma) was collected.

[0191] Isolation of single muscle cells Hindlimb muscles (i.e., gastrocnemius, EDL, tibialis anterior, and quadriceps) from wild-type and dystrophic mice were dissected and processed as previously described to obtain mononuclear cells. Muscles were washed with wash medium (Ham's F-10 supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin), added to muscle dissociation buffer (700-800 U / ml collagenase II (Worthington Biochemical Corporation) prepared in Ham's F-10 supplemented with 1% L-glutamine and 1% penicillin-streptomycin) (16 ml / hindlimb, 8 ml / diaphragm), minced with scissors, and incubated in a 37°C water bath with agitation (70 rpm) for 40 min. After centrifugation of the incubation samples at 500g for 5 min, dispase (11 U / ml, GIBCO) and collagenase II (235 U / mg) (1.5 ml / hindlimb, 0.5 ml / diaphragm) were added to induce enzymatic digestion, and the pellet was resuspended and triturated with a 10 ml serological pipette. The samples were agitated (70 rpm) for 20 min in a 37°C water bath, and then the cell suspension was mechanically dissociated into single cells by passing it through 18 and 19 gauge needles using a 10 ml syringe. The samples were centrifuged at 500g for 10 min at 4°C, the pellet was resuspended, filtered through a 40 μm nylon cell strainer (Euroclone), centrifuged (500g, 10 min at 4°C), and then resuspended in wash medium. The cells were incubated with primary antibodies for 45 min at 4°C on a rotating wheel (10 rpm) to label macrophages, fibrolipid-producing progenitor cells, and satellite cells. The list of primary antibodies used is summarized in Table 2. Samples were washed, APC streptavidin (1:100, BioLegend) was added, and samples were incubated for 20 min at 4° C. on a rotating wheel (10 rpm), then washed and resuspended in sorting buffer (washing medium + PBS with 1.5 mM EDTA, 2% BSA, 1% L-glutamine, and 1% penicillin-streptomycin, 1:1 ratio). Samples were finally filtered through cell strainer cap tubes (Thermo Fisher Scientific). [Table 4]

[0192] Cell populations were separated using a FACS ARIA™ II cell sorter (BD Biosciences). Physical parameters as forward scatter (FSC) and side scatter (SSC) were used to exclude cell clumps, debris, dead cells, and to isolate single cells. Satellite cells were purified by negative selection with anti-CD31, anti-CD45, anti-Sca1 antibodies and positive selection with anti-Vcam antibody, fibroadipogenic precursors were purified by negative selection with anti-CD31 and anti-CD45 antibodies and positive selection with anti-Sca1 antibody, and macrophages were purified by positive selection with anti-CD45 and anti-F4 / 80 antibodies. In samples taken from Pax7CreER / WT;R26RYFP / WT;mdx4Cv mice, satellite cells were purified by isolating the YFP+ve cell population. After sorting, cells were processed for RNA extraction and RT-PCR.

[0193] Real-time PCR Muscles were dissected and flash frozen prior to RNA extraction. Flash frozen muscles were homogenized using a mortar and pestle on dry ice under liquid nitrogen. Total RNA was extracted from muscles and FACS isolated cells using TRIzol® Reagent (Invitrogen) according to the manufacturer's instructions. RNA was quantified using a NanoDrop spectrophotometer and reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Gene expression was measured by quantitative RT-PCR using SYBR Green Master Mix (Thermo Fisher Scientific) and a C1000 Touch thermocycler-CFX96 Real Time System (Biorad). The thermo protocol used for RT-PCR is shown in Table 3. Primers spanning exon-exon junctions were used (Table 4). The levels of each transcript were measured using mouse HPRT (hypoxanthine-guanine phosphoribosyltransferase) mRNA levels as a standard. [Table 5] [Table 6]

[0194] Creatine kinase test C1qaKO;mdx, approximately 3 months old 4Cv and Pax7 CreER ;R26R YFP ;mdx 4Cv Serum samples taken from the mice were subjected to creatine kinase assay before sacrifice. A creatine kinase activity assay kit (colorimetric method) (Abcam, 155901) was used for the analysis according to the manufacturer's instructions.

[0195] Immunofluorescence Muscle sections were processed for immunofluorescence as known in the art. Briefly, dissected muscles were fixed with 0.5% paraformaldehyde for 4 hours, transferred to 30% sucrose overnight, frozen in optimal cutting temperature compound (OCT), and cryosectioned at 8 μm. The acquisition was performed using a Zeiss Axio Observer Z1 light microscope equipped with a monochrome camera (AxioCam 503 mono D). Anti-Axin 2 (ab32197, 1:20) and anti-C1q (ab11861, 1:50) were used as primary antibodies. Alexa Fluor 488 / 594 (Thermo Fisher Scientific) was used as secondary antibody.

[0196] Zen2 software (Zeiss) was used for immunofluorescence analysis. 2 The mean pixel intensity of C1q and Axin2 was measured for each biological replicate in ≥10 randomly selected areas. Background pixel intensity measured in sections stained only with secondary antibodies was subtracted. Normal or non-normal dataset distribution, Spearman (r), and Pearson coefficient (r) were determined using GraphPad Prism software.

[0197] statistical analysis Unless otherwise stated, the experiments presented herein were repeated at least three times. Data are presented as mean ± SEM unless otherwise stated. Statistical analysis was performed using GraphPad Prism 8. One-way ANOVA test was performed for multiple comparisons, and parametric Student's t test was performed for comparison between two groups. The number of biological replicates and the use of the specific test are indicated in the legend of each figure. Statistical significance was expressed as p-value (p): p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****.

[0198] Tissue lysis Brains (without olfactory bulbs and cerebellum) were lysed in 1:10 w / v BupH™ Tris-buffered saline (Thermo Scientific 28379) + protease inhibitor cocktail (Thermo Scientific A32963) + 10 mM EDTA) by homogenization with 7 mm steel beads in a Qiagen TissueLyser for 2 min at 30 Hz. Lysates were then spanned at 17,000 x g for 20 min. Supernatants were used for ELISA assays. Total protein was measured using the Pierce™ BCA Protein Assay Kit (ThermoFisher 23225).

[0199] PK and complement analysis Levels of free anti-C1q blocking mouse antibodies, free C1q, total C1q, C1s, C4, C2, C3, and activation markers C1q-C3d complex, C1s-C1inh complex, and C3d were measured in plasma and tissue lysates using sandwich ELISA. Black 96-well plates (Costar #3925) were coated with 75 μL of each capture antibody (Table 5) in bicarbonate buffer (pH 9.4) overnight at 4C. The next day, the plates were washed with dPBS pH 7.4 (Dulbecco's Phosphate Buffered Saline) and then blocked with dPBS buffer containing 3% bovine serum albumin (BSA). Standard curves were made with purified proteins in assay buffer (dPBS containing 0.3% BSA, 0.1% Tween 20, 10 mM EDTA). Serum or plasma samples for testing were prepared in assay buffer at the respective dilutions. The blocking buffer was removed from the plates by tapping. Standards and samples were added in duplicate at 75 μL per well and incubated with shaking at 300 rpm for 1 hour at room temperature for PK measurements, followed by overnight incubation at 4C for other assays. Plates were washed 3 times with wash buffer (dPBS containing 0.05% Tween 20) and 75 μL of alkaline phosphatase-conjugated secondary antibody (Table 5) was added to all wells. Plates were incubated 1 hour with shaking at room temperature. Plates were washed 3 times with wash buffer and developed using 75 μL of alkaline phosphatase substrate (Life Technologies, T2214). After 20 minutes at room temperature, plates were read using a luminometer. Standards were fitted using a 4PL logistic fit to determine unknown concentrations. Analyte levels were corrected for dilution and then plotted using GraphPad Prism. [Table 7]

[0200] Example 2: Evaluation of complement levels in skeletal muscle of wild-type, dystrophic and aged wild-type mice Levels of classical complement proteins C1q, C3, and C3d were measured in wild-type mice aged approximately 1 year, wild-type mice aged approximately 2 years, and mdx mice aged approximately 1 year. 4Cv Mice were assessed via ELISA in diaphragm, quadriceps and anterior tibia.

[0201] Figure 1: mdx compared with age-matched controls (approximately 1 year old) 4Cv Increased levels of C1q, C3 and C3d were observed in the anterior tibia of mice, and increased levels of C1q were observed in the anterior tibia of mice aged approximately 2 years compared to wild type mice aged approximately 1 year. 4Cv A trend was observed suggesting increased levels of C1q, C3 and C3d in the diaphragm of mice. C1q levels were significantly increased in mdx mice compared to age-matched controls (approximately 1 year old). 4Cv Finally, a trend suggesting increased C1q and C3 levels was observed in the quadriceps of mice aged approximately 2 years compared to wild-type mice aged approximately 1 year, with both C3 and C3d levels showing a similar trend.

[0202] Levels of classical complement proteins C1q, C1s, C3, C3d, and C4 were measured in wild-type and mdx mice aged approximately 1 month, 3 months, and 1 year. 4Cv Mdx mice were assessed via ELISA in the diaphragm, quadriceps and anterior tibia of 100 mice. Importantly, mice were perfused with PBS prior to muscle dissection and ELISA in order to limit the assessment to muscle and exclude serum protein components from the analysis. C1q, C1s, C3, C3d and C4 were expressed in all wild-type and mdx mice analyzed. 4Cv Expressed in muscle.

[0203] Figures 2 and 3: C1q, C1s, C3 and C3d protein expression was higher in dystrophic muscles of 1 month, 3 months and 1 year old compared to wild type in diaphragm, quadriceps and tibia anterior, reaching statistical significance in most cases. C4 protein expression was higher in dystrophic tibia anterior and quadriceps of 1 month old mice compared to wild type, in diaphragm of 3 month old dystrophic mice compared to wild type, and in tibia anterior of 1 year old dystrophic mice compared to wild type.

[0204] Example 3: Behavioral studies in wild-type and dystrophic mice Behavioral testing was performed on the same wild-type and dystrophic ∼1-month-old, ∼3-month-old, and ∼1-year-old mice analyzed for complement protein expression ( Figs. 2 and 3 ). The following functional parameters were evaluated: 1) maximum hanging time before fatigue, assessed by the four-limb hanging wire test; 2) The total distance traveled, the average speed, and the percentage of time traveled were assessed by open field test.

[0205] Figure 4: Wild-type and dystrophic mice showed differential muscle resistance in behavioral tests and ELISAs performed prior to muscle dissociation, consistent with reported motor deficits occurring in mdx mice compared to healthy controls. Dystrophic mice had lower resistance in the four-limb hanging wire test compared to age-matched wild-types at 3 months and 1 year of age. In the open field test, dystrophic mice at 3 months of age walked shorter distances at a lower speed and spent more immobile time compared to wild-types, but no differences were observed between dystrophic mice at 1 month of age and 1 year of age compared to age-matched wild-types.

[0206] Example 4: Evaluation of the efficacy of anti-C1q blocking antibodies in vivo Pax7 CreER ;R26R YFP ;mdx 4CvMice were treated with anti-C1q blocking antibody or control antibody. Behavioral tests were performed before and after treatment to assess functional parameters (i.e., maximum time before fatigue and motor activity). Blood samples were taken before and after treatment to assess complement and creatine kinase levels. After sacrifice, tissues (i.e., hind limb muscle, diaphragm, liver, and heart) were harvested for complement level and gene expression analysis. The experimental design used for anti-C1q blocking antibody treatment is shown in Figure 5.

[0207] Behavioral testing and sample (i.e., blood and tissue) collection were performed using C1qa expression conditionally ablated in myeloid cell lines. KO ;mdx 4Cv It was also performed in mice. KO ;mdx 4Cv Validation of the mice is shown in FIG.

[0208] Example 5: C1qa treated with anti-C1q blocking antibody KO ;mdx 4Cv Behavioral studies in mice and dystrophic mice C1qa at approximately 1 month, 2 months, and 3 months of age KO ;mdx 4Cv Mice were subjected to behavioral testing to assess maximum hanging time before fatigue (via a four limb hanging wire test), total distance traveled, average speed, and percentage of time moved (via an open field test).

[0209] The same behavioral tests were performed on Pax7 CreER ;R26R YFP ;mdx 4Cv Mice were treated with anti-C1q blocking antibody or control antibody before and after treatment.

[0210] Figures 7-10: At approximately 1 month of age (i.e., the first day of testing on postnatal days 31-36), C1qa compared to controls in the four-limb hanging wire test. KO ;mdx 4CvWe observed a trend suggesting improved physical resistance in mice. The same trend was observed at approximately 2 months of age (i.e., first day of testing at 67-69 days of age), but was no longer observed when mice were tested at approximately 3 months of age (i.e., first day of testing at 79-81 days of age). At all time points tested (i.e., approximately 1 month, approximately 2 months, and approximately 3 months), C1qa was significantly improved compared to controls in the open field test. KO ;mdx 4Cv No differential locomotor activity was observed in the mice.

[0211] Dystrophic mice treated with anti-C1q blocking antibodies developed higher physical resistance in the four-limb hanging wire test compared to mice treated with control antibodies. No differences were observed in open field test parameters (i.e., total distance, average speed, and percentage of time traveled) in dystrophic mice treated with anti-C1q blocking antibodies compared to controls. Of note, behavioral tests were performed in these animals before and after treatment at approximately 2.5 months of age (i.e., the first day of testing on postnatal day 67) and approximately 3 months of age (i.e., the first day of testing on postnatal day 81), respectively (Figure 5).

[0212] Example 6: C1qa treated with anti-C1q blocking antibody KO ;mdx 4Cv Gene expression assessment of canonical Wnt target genes and fibrogenic genes in mice and dystrophic mice In this example, to assess whether C1q depletion resulted in a decrease in the expression of the canonical Wnt pathway and / or a decrease in the level of fibrosis, we analyzed C1qa cells treated with an anti-C1q blocking antibody. KO ;mdx 4Cv Mouse and Pax7 CreER ;R26R YFP ;mdx 4CvGene expression analysis was performed in muscle and single cells isolated from mice. The mRNA levels of canonical Wnt target genes (i.e., Tgfβ2, Lgr5, and Axin2) and fibrogenic genes (i.e., collagen 1a1, collagen 3a1, and fibronectin) were analyzed by C1qa blockade in mice treated with anti-C1q blocking antibody. KO ;mdx 4Cv Mouse and Pax7 CreER ;R26R YFP ;mdx 4Cv Mouse muscles (i.e., gastrocnemius and diaphragm) and isolated single cells (i.e., fibrotic / adipogenic progenitor cells and satellite cells) were evaluated.

[0213] Figures 11-14: Overall, compared to controls, C1qa KO ;mdx 4Cv No decreased expression of any of the Wnt target genes (ie, Tgfβ2, Lgr5 and Axin2) or fibrogenic genes (ie, collagen 1a1, collagen 3a1 and fibronectin) in muscle and single cells was observed.

[0214] In addition, Pax7 cells treated with anti-C1q blocking antibody were significantly increased in comparison to controls. CreER ;R26R YFP ;mdx 4Cv No reduced expression of any of the Wnt target genes (ie, Tgfβ2, Lgr5 and Axin2) or fibrogenic genes (ie, collagen 1a1, collagen 3a1 and fibronectin) was observed in mouse muscle and single cells.

[0215] FIG. 27 shows the results of staining with anti-C1q, anti-Axin2, and Hoechst antibodies in mdx mice approximately 1 year old. 4Cv Representative immunofluorescence of gastrocnemius muscle from rats (upper image). Scale bar (upper image). A positive correlation between C1q and Axin2 intensity values ​​in each region was demonstrated.

[0216] Example 7: C1qa KO ;mdx 4CvCreatine kinase assay in sera from mice and from dystrophic mice treated with anti-C1q blocking antibody Serum creatine kinase (CK) levels are commonly used as an indicator of muscle damage in dystrophic mice and as a diagnostic biomarker for DMD. To assess whether C1q depletion led to a decrease in CK levels, C1qa KO ;mdx 4Cv Mouse and Pax7 CreER ;R26R YFP ;mdx 4Cv CK activity was measured in mice treated with anti-C1q blocking antibodies. CK tests were performed in both cases using serum samples taken after sacrifice (approximately 3 months of age).

[0217] Figure 15: C1qa compared to controls KO ;mdx 4Cv A trend suggesting a decrease in CK activity in serum was observed.

[0218] In this example, Pax7 after treatment with C1q blocking antibody and control antibody CreER ;R26R YFP ;mdx 4Cv We observed a trend suggesting a decrease in CK activity in mice, however, the different CK activity levels measured in the same samples before treatment (Cntr(pre) and anti-C1q(pre), Figure 15B, likely similar) suggest biological variability.

[0219] Example 8: C1qa KO ;mdx 4Cv Assessment of complement levels in plasma and muscle from mice and dystrophic mice treated with anti-C1q blocking antibody C1qa KO ;mdx 4CvELISA assays were performed to assess complement levels in plasma and tissues from mice and dystrophic mice treated with anti-C1q blocking antibodies. Levels of classical complement proteins (i.e., C1q, C3d, and C1s), C1q-C3d immune complexes (IC), and C1s-C1 inhibitor complexes (C1sC1inh) were measured using the ELISA kit. KO ;mdx 4Cv The levels of the proteins were evaluated in plasma, diaphragm, gastrocnemius and liver from mice, as well as from dystrophic mice treated with anti-C1q blocking antibodies. The drug levels present in the samples were also evaluated in samples taken from dystrophic mice treated with anti-C1q blocking antibodies (PK). In addition, the levels of all the aforementioned proteins were evaluated in heart samples taken from dystrophic mice treated with anti-C1q blocking antibodies.

[0220] Figures 16-22: With regard to anti-C1q blocking antibody treatment, the drug levels found in all samples taken from mice treated with anti-C1q blocking antibody (i.e., plasma, diaphragm, gastrocnemius, liver and heart) were increased compared to mice treated with control antibody. Among the samples analyzed, gastrocnemius was the muscle with the highest amount of drug and heart was the muscle with the lowest amount of drug. The levels of C1q protein were reduced after treatment with anti-C1q blocking antibody in all analyzed samples. Notably, C1q depletion was strongly effective in diaphragm, gastrocnemius and liver, and C1q levels in samples taken from mice treated with anti-C1q blocking antibody were significantly higher than those in C1q genetically ablated mice (i.e., C1q KO ;mdx 4Cv ) were similar to the levels detected in samples taken from

[0221] Overall, there was a significant increase in the levels of other tested proteins (i.e., C3d, C1s) and protein complexes (i.e., IC, C1sC1inh) in samples taken from dystrophic mice treated with anti-C1q blocking antibodies compared to controls, as well as in C1q KO ;mdx 4CvNo differences were observed in the samples compared to the controls.

[0222] Example 9: Assessment of complement levels in wild-type and dystrophic skeletal muscle Levels of C1 complex subunits: C1qa, C1qb, C1qc, C1r, and C1s were measured in wild-type and mdx mice approximately 1 year of age. 4Cv Expression of C1qa and C1qb was assessed by measuring mRNA expression in hindlimb muscles. Cv was measured in macrophages isolated from hindlimb muscles.

[0223] Figures 23 and 26: Expression of C1 complex components is enhanced in dystrophic muscle. The mRNA expression of C1 complex subunits C1qa, C1qb, C1qc, C1r, and C1s was increased in wild-type (WT) and mdx mice approximately 1 year of age. 4Cv (Mdx) mice.

[0224] Figures 24A-24C: C1q subunits are expressed by infiltrating macrophages in skeletal muscle of dystrophic mice. Figures 24A-24B show qPCR analysis of C1qa (A) and C1qb (B) expression in satellite cells (SCs), macrophages (MACs) and fibrotic / adipogenic progenitor cells (FAPs) isolated from hind limb muscles of wild-type (WT) and mdxCv (Mdx) mice at approximately 1 year of age. Figure 24C shows qPCR analysis of C1qa (A) and C1qb (B) expression in satellite cells (SCs), macrophages (MACs) and fibrotic / adipogenic progenitor cells (FAPs) isolated from hind limb muscles of wild-type (WT) and mdxCv (Mdx) mice at approximately 1 year of age. 4Cv (Mdx) The number of macrophages per mg of tissue in hindlimb muscle of mice is shown.

[0225] Example 10: C1qa treated with anti-C1q blocking antibody KO ;mdx 4Cv Behavioral studies in mice and dystrophic mice C1qa KO ;mdx 4CvMice were subjected to behavioral testing at approximately 1 month, 2 months, and 3 months of age to assess maximum hanging time before fatigue (via the four-limb hanging wire test), total distance traveled, average speed, percentage of time traveled (via the open field test), two-limb grip strength, and rotating rod speed.

[0226] The same behavioral tests were performed on Pax7 CreER ;R26R YFP ;mdx 4Cv Mice were treated with anti-C1q blocking antibody or control antibody before and after treatment.

[0227] Figures 25A to 25I show C1qa KO ;mdx 4Cv Behavioral testing in mice and controls. Cre+ / - C1qa FL / FL ;mdx 4Cv (C1qaKO) and Lyz Cre+ / - C1qa WT / WT ;mdx 4Cv(CNTR) Mouse weight (grams) is shown. Figures 25B, 25C show hanging test (HT) performed on mice as in (Figure 25A). Total hanging time (Figure 25B) and total hanging time normalized for mouse weight (Figure 25C) were evaluated. Figures 25D-25F show open field (OF) test performed on mice as in (Figure 25A). Total distance (cm) (Figure 25D), average speed (cm / sec) (Figure 25E) and percentage of moving time (Figure 25F) were evaluated. Figures 25G, 25H show two leg grip test performed on mice as in (Figure 25A). Maximum strength normalized for mouse weight (Figure 25G) and total grip time normalized for mouse weight (Figure 25H) were evaluated. Figure 25I shows rotarod test performed on mice as in (Figure 25A). Total walking time normalized for mouse weight was evaluated. N=5 (C1qaKO), N=7 (CNTR), N=2 (WT). Data are expressed as mean with SEM. Two-tailed unpaired t-test was applied. p>0.05: ns; p≦0.05: *; p≦0.01: **; p≦0.001: *** and p≦0.0001: ****.

[0228] Incorporation by Reference Each of the patents, published patent applications, and non-patent publications cited herein is hereby incorporated by reference in its entirety.

[0229] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A composition for preventing, reducing the risk of developing, delaying or blocking the progression of, or treating Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD), type VI collagen-related disorders, congenital muscular dystrophy (CMD) or congenital myopathy, or distal muscular dystrophy / myopathy, comprising an inhibitor of the classical complement pathway.

2. 2. The composition of claim 1, wherein the LGMD is a sarcoglycanopathy, dystroglycanopathy, or dysferlinopathy; the type VI collagen-related disorder is Bethlem myopathy or Ullrich congenital muscular dystrophy (UCMD); or the distal muscular dystrophy / myopathy is Miyoshi myopathy.

3. The composition of claim 1 , wherein the inhibitor of the classical complement pathway is a C1 complex inhibitor, a C1q inhibitor, a C1r inhibitor, or an anti-C1s antibody.

4. 4. The composition of claim 3, wherein the inhibitor is an antibody, peptide, protein, nucleic acid, small molecule, gene editor, base editor, or epigenetic editor, and optionally the nucleic acid is an antisense oligonucleotide, miRNA, miRNA inhibitor, mRNA, aptamer, or antisense nucleic acid.

5. The composition of claim 4 , wherein the antibody is an anti-C1q antibody or an antigen-binding fragment thereof.

6. The composition of claim 5, wherein the anti-C1q antibody or its antigen-binding fragment inhibits the interaction between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s, or the anti-C1q antibody promotes clearance of C1q from the circulation or tissues.

7. The anti-C1q antibody or antigen-binding fragment thereof has a dissociation constant (K Ｄ 6. The composition of claim 5, wherein the anti-C1q antibody or antigen-binding fragment thereof binds to C1q with a binding stoichiometry in the range of 20:1 to 1.0:1, 6:1 to 1.0:1, 2.5:1 to 1.0:1, or less than 1.0:

1.

8. The anti-C1q antibody or antigen-binding fragment thereof specifically binds to C1q and neutralizes its biological activity, and optionally the biological activity is (a) (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to IgM, (5) C1q binding to phosphatidylserine, (6) C1q binding to pentraxin-3, (7) C1q binding to C-reactive protein (CRP), (8) C1q binding to globular C1q receptor (gC1qR), (9) C1q binding to complement receptor 1 (CR1), (10) C1q binding to beta-amyloid, (11) C1q binding to calreticulin, (12) C1q binding to apoptotic cells, or (13) C1q binding to B cells, or (b) The composition of claim 5, wherein the induction of (1) activation of the classical complement activation pathway, (2) activation of antibody and complement-dependent cytotoxicity, (3) CH50 hemolysis, (4) synapse loss, (5) B cell antibody production, (6) dendritic cell maturation, (7) T cell proliferation, (8) cytokine production, (9) microglial activation, (10) immune complex formation, (11) phagocytosis of synapses or nerve terminals, (12) activation of complement receptor 3 (CR3 / C3)-expressing cells, or (13) neuroinflammation.

9. The composition of claim 8, wherein the CH50 hemolysis comprises human CH50 hemolysis, and optionally the anti-C1q antibody or antigen-binding fragment thereof is capable of neutralizing at least about 50% to about 100% of the human CH50 hemolysis.

10. The composition of claim 5, wherein the anti-C1q antibody or its antigen-binding fragment is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a monovalent antibody, a multispecific antibody, or an antibody derivative thereof, or the anti-C1q antibody is an antigen-binding fragment, and the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule.

11. The composition of claim 5, wherein the anti-C1q antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO: 7, and / or a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO:

11.

12. The composition of claim 11, wherein the anti-C1q antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an amino acid sequence having at least about 95% identity to an amino acid sequence selected from SEQ ID NOs: 4 and 35 to 38, preferably wherein the light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 4 and 35 to 38.

13. The composition of claim 11 or 12, wherein the anti-C1q antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% identity to an amino acid sequence selected from SEQ ID NOs: 8 and 31 to 34, preferably wherein the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 8 and 31 to 34.

14. 6. The composition of claim 5, wherein the antigen-binding fragment comprises a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO:

40.

15. The composition of claim 3 or 4, wherein the inhibitor of the classical complement pathway is a C1s inhibitor.

16. The composition of claim 4 , wherein the antibody is an anti-C1s antibody or an antigen-binding fragment thereof.

17. The anti-C1s antibody or antigen-binding fragment thereof has a dissociation constant (K Ｄ 17. The composition of claim 16, wherein

18. A composition for preventing, reducing the risk of developing, slowing or blocking the progression of, or treating Duchenne muscular dystrophy, comprising an inhibitor of the classical complement pathway.

19. A composition for preventing, reducing the risk of developing, delaying or blocking the progression of, or treating Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD), type VI collagen-related disorder, congenital muscular dystrophy (CMD) or congenital myopathy, or distal muscular dystrophy / myopathy, comprising an anti-C1q antibody or antigen-binding fragment thereof, wherein the anti-C1q antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO:

11.

20. Use of an inhibitor of the classical complement pathway in the manufacture of a medicament for preventing, reducing the risk of developing, delaying or blocking the progression of, or treating Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD), type VI collagen-related disorders, congenital muscular dystrophy (CMD) or congenital myopathy, or distal muscular dystrophy / myopathy.

21. Use of an inhibitor of the classical complement pathway in the manufacture of a medicament for preventing, reducing the risk of developing, slowing or blocking the progression of, or treating Duchenne muscular dystrophy.

22. Use of an anti-C1q antibody or antigen-binding fragment thereof in the manufacture of a medicament for preventing, reducing the risk of onset, delaying or blocking the progression of, or treating Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD), type VI collagen-related disorder, congenital muscular dystrophy (CMD) or congenital myopathy, or distal muscular dystrophy / myopathy, wherein the anti-C1q antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO: 11.

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