Patent Application
20220143136
This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 2018-119R_Seqlisting.txt; Size: 168,826 bytes; Created: Dec. 20, 2019), which is incorporated by reference in its entirety.
Plasma membrane repair occurs after membrane disruption and is a highly conserved process. The active process required for resealing membrane disruptions is thought to rely on Ca2+-dependent vesicle fusion and local cytoskeletal remodeling (McNeil and Khakee, 1992; McNeil and Kirchhausen, 2005). Other models suggest that membrane repair is mediated through the fusion of lysosomal vesicles and/or lateral diffusion of membrane to the site of injury (Demonbreun et al., 2016b; McDade et al., 2014; Reddy et al., 2001; Rodriguez et al., 1997). These models are not mutually exclusive and may depend on the type and extent of damage. Skeletal muscle is highly dependent on plasma membrane repair as mutation in genes encoding repair proteins lead to muscle disease (Bansal et al., 2003; Bashir et al., 1998; Cai et al., 2009; Defour et al., 2017; Demonbreun and McNally, 2016; Demonbreun et al., 2015).
Annexins are Ca2+-binding proteins that regulate lipid binding, cytoskeletal reorganization, and bleb formation, steps necessary for membrane repair (Bizzarro et al., 2012; Boye et al., 2018; Boye et al., 2017; Grewal et al., 2017; Jimenez and Perez, 2017; Lauritzen et al., 2015). Individual annexin repeat domains coordinate Ca2+ binding with unique annexin-specific type II or type Ill binding sites. Differential Ca2+ affinity of the type II and type Ill binding sites provides each annexin a unique ability to respond to a range of intracellular Ca2+ levels and phospholipid binding (Blackwood and Ernst, 1990). Annexins have the ability to self- and hetero-oligomerize (Zaks and Creutz, 1991). Typical annexins like A1 and A2 contain one annexin core composed of four annexin repeat domains. In contrast, annexin A6 contains two annexin cores and thus eight annexin repeat domains (Benz et al., 1996). Annexin A6's duplicated structure makes it possible for the amino- and carboxyl-terminal annexin core domains to bind one or two distinct membranes making annexin A6 a prime target for facilitating membrane coalescence and folding required during membrane repair (Boye et al., 2018; Boye et al., 2017; Buzhynskyy et al., 2009).
Annexins have a high affinity for phosphatidylserine, phosphatidylinositol, and cholesterol, which are highly enriched in the sarcolemma (Fiehn et al., 1971; Gerke et al., 2005). Multiple annexins have been implicated in membrane repair in skeletal muscle, as well as Xenopus oocytes, human trophoblasts, and HeLa cancer cells, suggesting a conserved mechanism (Babbin et al., 2008; Bement et al., 1999; Carmeille et al., 2015; Davenport et al., 2016; Demonbreun et al., 2016b; Lennon et al., 2003; McNeil et al., 2006; Roostalu and Strahle, 2012). Annexins are recruited to the injured membrane in a sequential manner forming a macromolecular repair complex at the membrane lesion (Boye et al., 2017; Demonbreun et al., 2016b; Roostalu and Strahle, 2012).
Defects in the ability of a cell to repair the plasma membrane lead to numerous diseases, including muscular dystrophy and other pathological conditions that result in cellular death. Membrane injury can result from events such as overuse, trauma, burn, chemical exposure and chronic disease. Currently, there is a lack of agents that prevent or treat membrane damage (Demonbreun and McNally, 2016). Annexins are calcium-binding proteins that have a high affinity for membrane lipids. Annexins have been implicated in membrane repair in many cell types including muscle, trophoblasts, HeLa cells, and oocytes, suggesting a widespread role for annexins (Babbin et al., 2008; Bement et al., 1999; Carmeille et al., 2015; Davenport et al., 2016; Demonbreun et al., 2016b; McNeil et al., 2006; Roostalu and Strahle, 2012). Annexin A1 (“A1”), annexin A2 (“A2”), annexin A5 (“A5”), and annexin A6 (“A6”) are sequentially recruited to the injury site forming a large repair complex (Demonbreun et al., 2016b; Roostalu and Strahle, 2012). A polymorphism in Anxa6 was identified that created a truncated form of annexin A6 (Swaggart et al., 2014). Expression of truncated annexin A6 acted in a dominant-negative fashion, decreasing annexin repair complex formation. This correlated with impaired membrane repair and more severe muscle disease (Demonbreun et al., 2016a; Demonbreun et al., 2016b; Quattrocelli et al., 2017b; Swaggart et al., 2014). Conversely, overexpression of annexin A6 was sufficient to improve repair and decrease susceptibility to membrane injury (Quattrocelli et al., 2017a). Moreover, treatment with recombinant annexin A6 was sufficient to reduce muscle damage in cell based and whole animal studies.
An aspect of the disclosure is drawn to a method of treating a cellular membrane injury comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein. Another aspect of the disclosure is directed to a method of delaying onset, enhancing recovery from cellular membrane injury, or preventing a cellular membrane injury comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein. Embodiments of each of these aspects are contemplated wherein the agent is selected from the group consisting of a recombinant protein, a steroid, and a polynucleotide capable of expressing an annexin protein. In some embodiments, the steroid is a corticosteroid or a glucocorticoid. In some embodiments, the recombinant protein is an annexin protein. In some embodiments, the annexin protein is annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, the recombinant protein is a wild type annexin protein, a modified annexin protein, an annexin-like protein, or a fragment of a wild type annexin protein or annexin-like protein. In some embodiments, the modified annexin protein is annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, the patient suffers from an acute injury. In some embodiments, the acute injury results from surgery, a burn, a toxin, a chemical, radiation-induced injury, acute myocardial injury, acute muscle injury, acute lung injury, acute epithelial injury, acute epidermal injury, acute kidney injury, acute liver injury, vascular injury, an excessive mechanical force, or trauma. In some embodiments, the patient suffers from a chronic disorder. In some embodiments, the patient suffers from a chronic disorder, such as Becker Muscular Dystrophy (BMD), Duchenne Muscular Dystrophy (DMD), Limb Girdle Muscular Dystrophy, congenital Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy (EDMD), Myotonic Dystrophy, Fascioscapulohumeral Dystrophy (FSHD), Oculopharyngeal Muscular Dystrophy, Distal Muscular Dystrophy, cystic fibrosis, pulmonary fibrosis, muscle atrophy, cerebral palsy, an epithelial disorder, an epidermal disorder, a kidney disorder, a liver disorder, sarcopenia, or cardiomyopathy (hypertrophic, dilated, congenital, arrhythmogenic, restrictive, ischemic, heart failure). In some embodiments, the cardiomyopathy is hypertrophic, dilated, congenital, arrhythmogenic, restrictive, ischemic, or heart failure.
In some embodiments, the methods disclosed herein further comprise administering an effective amount of a second agent, wherein the second agent is selected from the group consisting of mitsugumin 53 (MG53), a modulator of latent TGF-β binding protein 4 (LTBP4), a modulator of transforming growth factor β (TGF-β) activity, a modulator of androgen response, a modulator of an inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and a combination thereof.
In some embodiments of the methods disclosed herein, the polynucleotide is associated with a nanoparticle. In some embodiments, the polynucleotide is contained in a vector. In some embodiments, the vector is within a chloroplast. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of a herpes virus vector, an adeno-associated virus (AAV) vector, an adeno virus vector, and a lentiviral vector. In some embodiments, the AAV vector is recombinant AAV5, AAV6, AAV8, AAV9, or AAV74, including embodiments wherein the AAV74 vector is AAVrh74.
In some embodiments of the methods disclosed herein, the composition increases the activity of annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18), or a combination thereof. In some embodiments, the composition increases the activity of annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, the composition increases the activity of annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3) and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, the composition increases the activity of annexin A1 (SEQ ID NO: 1) and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, the composition increases the activity of annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In any of the aspects or embodiments of the disclosure, the composition for use in any of the methods described herein is a pharmaceutical composition as disclosed herein.
In some aspects, the disclosure provides a pharmaceutical composition comprising a modified annexin protein and a pharmaceutically acceptable carrier, buffer, and/or diluent. In some aspects, the disclosure provides a pharmaceutical composition comprising an annexin protein and a pharmaceutically acceptable carrier, buffer, and/or diluent. In some embodiments, the annexin protein or modified annexin protein is annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18), or a combination thereof. In some embodiments, the annexin protein or modified annexin protein is annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In further embodiments, the pharmaceutical composition comprises annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, the pharmaceutical composition comprises annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3) and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In further embodiments, the pharmaceutical composition comprises annexin A1 (SEQ ID NO: 1) and annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, the pharmaceutical composition further comprises a steroid. In further embodiments, the steroid is a corticosteroid or a glucocorticoid. In some embodiments, the pharmaceutical composition further comprises an effective amount of a second agent, wherein the second agent is selected from the group consisting of mitsugumin 53 (MG53), a modulator of latent TGF-β binding protein 4 (LTBP4), a modulator of transforming growth factor β (TGF-β) activity, a modulator of androgen response, a modulator of an inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and a combination thereof. In further embodiments, purity of the annexin protein in the composition is about 90% or higher as measured by standard release assay, including but not limited to SDS-PAGE, SEC-HPLC, and immunoblot analysis. In some embodiments, the composition has an endotoxin level that is less than about 0.50000 endotoxin units per milligram (EU/mg) A280 annexin protein. In some embodiments, the modified annexin protein is produced in a prokaryotic cell.
In some aspects, the disclosure provides a pharmaceutical composition comprising modified annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof) and a pharmaceutically acceptable carrier, buffer, and/or diluent. In some aspects, the disclosure provides a pharmaceutical composition comprising annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof) and a pharmaceutically acceptable carrier, buffer, and/or diluent. In some embodiments, the pharmaceutical composition further comprises annexin A1 (SEQ ID NO: 1) and annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3). In some embodiments, the pharmaceutical composition further comprises annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3). In some embodiments, the pharmaceutical composition further comprises annexin A1 (SEQ ID NO: 1). In some embodiments, the pharmaceutical composition further comprises a steroid. In further embodiments, the steroid is a corticosteroid or a glucocorticoid. In some embodiments, the pharmaceutical composition further comprises an effective amount of a second agent, wherein the second agent is selected from the group consisting of mitsugumin 53 (MG53), a modulator of latent TGF-β binding protein 4 (LTBP4), a modulator of transforming growth factor β (TGF-β) activity, a modulator of androgen response, a modulator of an inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and a combination thereof. In some embodiments, purity of the annexin protein in the composition is about 90% or higher as measured by standard release assay. In some embodiments, the composition has an endotoxin level that is less than about 0.50000 endotoxin units per milligram (EU/mg). In some embodiments, the modified annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof) is produced in a prokaryotic cell.
Other features and advantages of the disclosure will be better understood by reference to the following detailed description, including the figures and the examples.
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee.
Membrane repair is essential to cell survival and requires a quick and efficient process to limit cellular injury. Muscle is prone to membrane disruption due to the elongated shape of muscle cells and the continuous stress brought on by muscle contractions. Mutations in genes encoding membrane-associated proteins produce fragile membranes, reduce membrane repair capacity and result in progressive muscular dystrophy. Therapeutics and methods disclosed herein to enhance membrane repair and reduce susceptibility to membrane injury are contemplated to benefit muscle in both acute and chronic injury settings.
As used herein, an agent that “increases the activity of an annexin protein” is one that increases a property of an annexin protein as a calcium-binding membrane associated repair protein that enhances restoration of membrane integrity. The enhancement to restoring membrane integrity may be through facilitating the formation of a macromolecular repair complex at the membrane lesion including proteins such as, without limitation, annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), EHD2, dysferlin, and MG53.
As used herein, the term “treating” or “treatment” refers to an intervention performed with the intention of preventing the further development of or altering the pathology of a disease or infection. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Of course, when “treatment” is used in conjunction with a form of the separate term “prophylaxis,” it is understood that “treatment” refers to the narrower meaning of altering the pathology of a disease or condition. “Preventing” refers to a preventative measure taken with a subject not having a condition or disease. A therapeutic agent may directly decrease the pathology of a disease, or render the disease more susceptible to treatment by another therapeutic agent(s) or, for example, the host's own cellular membrane repair system. Treatment of patients suffering from clinical, biochemical, or subjective symptoms of a disease may include alleviating one or more of such symptoms or reducing the predisposition to the disease. Improvement after treatment may be manifested as a decrease or elimination of one or more of such symptoms.
The annexin protein family is characterized by the ability to bind phospholipids and actin in a Ca2+-dependent manner. Annexins preferentially bind phosphatidylserine, phosphatidylinositols, and cholesterol (Gerke et al., 2005). In humans, dominant or recessive mutations in annexin genes have not been associated with muscle disease. However, annexin A5 genetic variants are associated with pregnancy loss (de Laat et al., 2006). The annexin family is known to comprise over 160 distinct proteins that are present in more than 65 unique species (Gerke and Moss, 2002). Humans have 12 different annexin genes, characterized by distinct tissue expression and localization. Annexins are involved in a variety of cellular processes including membrane permeability, mobility, vesicle fusion, and membrane bending. These properties are Ca2+-dependent. Although annexins do not contain EF hand domains, calcium ions bind to the individual annexin repeat domains. Differential Ca2+ affinity allows each annexin protein to respond to changes in intracellular calcium levels under unique spatiotemporal conditions (Blackwood and Ernst, 1990).
Structurally, the annexin family of proteins contains a conserved carboxy-terminal core domain composed of multiple annexin repeats and a variable amino-terminal head. The amino-terminus differs in length and amino acid sequence amongst the annexin family members. Additionally, post-translational modifications alter protein function and protein localization (Goulet et al., 1992; Kaetzel et al., 2001). Annexin proteins have the potential to self-oligomerize and interact with membrane surfaces and actin in the presence of Ca2+ (Zaks and Creutz, 1991, Hayes et al., 2006), Jaiswal et al., 2014)). The amino-terminal region is thought to bind actin or one lipid membrane in a Ca2+-dependent manner, while the annexin core region binds an additional lipid membrane.
Annexins do not contain a predicted hydrophobic signal sequence targeting the annexins for classical secretion through the endoplasmic reticulum, yet annexins are found both on the interior and exterior of the cell (Christmas et al., 1991; Deora et al., 2004; Wallner et al., 1986). The process by which the annexins are externalized remains unknown. It is hypothesized that annexins may be released through exocytosis or cell lysis, although the method of externalization may vary by cell type. Functionally, localization both inside and outside the cell adds to the complexity of the roles annexins play within tissues and cell types. Annexin A5 is used commonly as a marker for apoptosis due to its high affinity to phosphatidylserine (PS). During cell death and injury, PS reverses membrane orientation from the inner to outer membrane, providing access for annexin binding from the cell exterior. Annexins have been shown to have anti-inflammatory, pro-fibrinolytic, and anti-thrombotic effects. The annexin A1-deleted mouse model exhibits an exacerbated inflammatory response when challenged and is resistant to the anti-inflammatory effects of glucocorticoids (Hannon et al., 2003). The annexin A2 null-mouse develops fibrin accumulation in the microvasculature and is defective in clearance of arterial thrombi (Ling et al., 2004). Although little is known about the precise function of extracellular annexins, the expression level of annexin proteins may function as a diagnostic marker for a number of diseases due to the strong correlation between high expression levels of annexins and the clinical severity of disease (Cagliani et al., 2005).
In some aspects, the disclosure provides methods of the disclosure contemplate treating a cellular membrane injury comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein. In further aspects, methods of delaying onset, preventing a cellular membrane injury, or enhancing recovery from cellular membrane injury are provided, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising an agent that increases the activity of an annexin protein. “Increase the activity of an annexin protein” means that administration of the agent results in an overall increase in the activity (i.e., the increase in activity derived from administration of the agent plus any endogenous activity) of one or more annexin proteins as disclosed herein.
The term “agent” as used herein refers to a recombinant protein (e.g., a recombinant annexin protein), a steroid, an annexin peptide, and a polynucleotide capable of expressing an annexin protein.
Methods of the disclosure include those in which a recombinant protein (e.g., one or more annexin proteins) is administered to a patient in need thereof in a therapeutically effective amount. Thus, in any of the aspects or embodiments of the disclosure, the agent that increases the activity of an annexin protein is a recombinant protein (e.g., an annexin protein). As used herein a “protein” refers to a polymer comprised of amino acid residues. “Annexin protein” as used herein includes without limitation a wild type annexin protein, a modified annexin protein, an annexin-like protein, or a fragment, analog, variant, fusion or mimetic, each as described herein. An “annexin peptide” is a shorter version (e.g., about 50 amino acids or less) of a wild type annexin protein, an annexin-like protein, or a fragment, analog, variant, fusion or mimetic that is sufficient to increase the overall activity of the annexin protein to which the annexin peptide is related.
Proteins of the present disclosure may be either naturally occurring or non-naturally occurring. Naturally occurring proteins include without limitation biologically active proteins that exist in nature or can be produced in a form that is found in nature by, for example, chemical synthesis or recombinant expression techniques. Naturally occurring proteins also include post-translationally modified proteins, such as, for example and without limitation, glycosylated proteins. Non-naturally occurring proteins contemplated by the present disclosure include but are not limited to synthetic proteins, as well as fragments, analogs and variants of naturally occurring or non-naturally occurring proteins as defined herein. Non-naturally occurring proteins also include proteins or protein substances that have D-amino acids, modified, derivatized, or non-naturally occurring amino acids in the D- or L-configuration and/or peptidomimetic units as part of their structure. The term “protein” typically refers to large polypeptides. The term “peptide” generally refers to short (e.g., about 50 amino acids or less) polypeptides.
Non-naturally occurring proteins are prepared, for example, using an automated protein synthesizer or, alternatively, using recombinant expression techniques using a modified oligonucleotide which encodes the desired protein.
As used herein a “fragment” of a protein is meant to refer to any portion of a protein smaller than the full-length protein expression product.
As used herein an “analog” refers to any of two or more proteins substantially similar in structure and having the same biological activity, but can have varying degrees of activity, to either the entire molecule, or to a fragment thereof. Analogs differ in the composition of their amino acid sequences based on one or more mutations involving substitution, deletion, insertion and/or addition of one or more amino acids for other amino acids. Substitutions can be conservative or non-conservative based on the physico-chemical or functional relatedness of the amino acid that is being replaced and the amino acid replacing it.
As used herein a “variant” refers to a protein or analog thereof that is modified to comprise additional chemical moieties not normally a part of the molecule. Such moieties may modulate, for example and without limitation, the molecule's solubility, absorption, and/or biological half-life. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art. In various aspects, polypeptides are modified by biotinylation, glycosylation, PEGylation, and/or polysialylation.
Fusion proteins, including fusion proteins wherein one fusion component is a fragment or a mimetic, are also contemplated. A “mimetic” as used herein means a peptide or protein having a biological activity that is comparable to the protein of which it is a mimetic.
In any of the aspects or embodiments of the disclosure, the recombinant protein is an annexin protein (e.g., a recombinant wild type annexin protein, a modified annexin protein, an annexin-like protein, or a fragment of a wild type annexin protein or annexin-like protein that exhibits one or more biological activities of an annexin protein). By “annexin-like protein” is meant a protein having sufficient amino acid sequence identity to a referent wild type annexin protein to exhibit the activity of an annexin protein, for example and without limitation, activity as a calcium-binding membrane associated repair protein that enhances restoration of membrane integrity through facilitating the formation of a macromolecular repair complex at the membrane lesion including proteins such as annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), EHD2, dysferlin, and MG53. In some embodiments, the annexin-like protein is a protein having about or at least about 75% amino acid sequence identity with a referent wild type human annexin protein (e.g., annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), or annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18)). In further embodiments, the annexin-like protein is a protein having about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, or about 99% amino acid sequence identity with a wild type human annexin protein.
In some embodiments, an agent of the disclosure is an annexin protein that comprises a post-translational modification. In various embodiments, the post-translational modification increases production of an annexin or annexin-like protein, increases solubility of an annexin or annexin-like protein, decreases aggregation of an annexin or annexin-like protein, increases the half-life of an annexin or annexin-like protein, increases the stability of an annexin or annexin-like protein, enhances target membrane engagement of an annexin or annexin-like protein, or is a codon-optimized version of an annexin or annexin-like protein.
In some embodiments, an agent of the disclosure is a polynucleotide capable of expressing an annexin protein as described herein. The term “nucleotide” or its plural as used herein is interchangeable with modified forms as discussed herein and otherwise known in the art. In certain instances, the art uses the term “nucleobase” which embraces naturally-occurring nucleotide, and non-naturally-occurring nucleotides which include modified nucleotides. Thus, nucleotide or nucleobase means the naturally occurring nucleobases A, G, C, T, and U. Non-naturally occurring nucleobases include, for example and without limitations, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N′,N′-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine, isoguanine, inosine and the “non-naturally occurring” nucleobases described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term “nucleobase” also includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., 1991, Angewandte Chemie, International Edition, 30: 613-722 (see especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which are hereby incorporated by reference in their entirety). In various aspects, polynucleotides also include one or more “nucleosidic bases” or “base units” which are a category of non-naturally-occurring nucleotides that include compounds such as heterocyclic compounds that can serve like nucleobases, including certain “universal bases” that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Universal bases include 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole), and optionally substituted hypoxanthine. Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.
Modified nucleotides are described in EP 1 072 679 and WO 97/12896, the disclosures of which are incorporated herein by reference. Modified nucleobases include without limitation, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified bases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., 1991, Angewandte Chemie, International Edition, 30: 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these bases are useful for increasing binding affinity and include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are, in certain aspects combined with 2′-O-methoxyethyl sugar modifications. See, U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,750,692 and 5,681,941, the disclosures of which are incorporated herein by reference.
Methods of making polynucleotides of a predetermined sequence are well-known. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solid-phase synthesis methods are preferred for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Polynucleotides and polyribonucleotides can also be prepared enzymatically via, e.g., polymerase chain reaction (PCR). Non-naturally occurring nucleobases can be incorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am. Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124:13684-13685 (2002).
In some embodiments, the agent that increases the activity of an annexin protein is a steroid. In further embodiments, the steroid is a corticosteroid, a glucocorticoid, or a mineralocorticoid. In still further embodiments, the corticosteroid is Betamethasone, Budesonide, Cortisone, Dexamethasone, Hydrocortisone, Methylprednisolone, Prednisolone, or Prednisone. In some embodiments, the corticosteroid is salmeterol, fluticasone, or budesonide.
In some embodiments, the steroid is an anabolic steroid. In further embodiments anabolic steroids, include, but are not limited to, testosterone or related steroid compounds with muscle growth inducing properties, such as cyclostanazol or methadrostenol, prohomones or derivatives thereof, modulators of estrogen, and selective androgen receptor modulators (SARMS).
An appropriate expression vector may be used to deliver exogenous nucleic acid to a recipient muscle cell in the methods of the disclosure. In order to achieve effective gene therapy, the expression vector must be designed for efficient cell uptake and gene product expression. In some embodiments, the vector is within a chloroplast. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of a herpes virus vector, an adeno-associated virus (AAV) vector, an adeno virus vector, and a lentiviral vector.
Use of adenovirus or adeno-associated virus (AAV) based vectors for gene delivery have been described [Berkner, Current Topics in Microbiol. and Imunol. 158: 39-66 (1992); Stratford-Perricaudet et al., Hum. Gene Ther. 1: 241-256 (1990); Rosenfeld et al., Cell 8: 143-144 (1992); Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-630 (1992)]. In various embodiments, the adeno-associated virus vector is AAV5, AAV6, AAV8, AAV9, or AAV74. In some embodiments, the adeno-associated virus vector is AAV9. In further embodiments, the adeno-associated virus vector is AAVrh74.
Specific methods for gene therapy useful in the context of the present disclosure depend largely upon the expression system employed; however, most methods involve insertion of coding sequence at an appropriate position within the expression vector, and subsequent delivery of the expression vector to the target muscle tissue for expression.
Additional delivery systems useful in the practice of the methods of the disclosure are discussed in U.S. Patent Publication Numbers 2012/0046345 and 2012/0039806, each of which is incorporated herein by reference in its entirety.
In various aspects, the disclosure provides compositions for treating, delaying onset, enhancing recovery from, or preventing a cellular membrane injury, comprising administering an agent and optionally an additional agent to a patient in need thereof.
Such a patient is one that is suffering from, for example, Duchenne Muscular Dystrophy, Limb Girdle Muscular Dystrophy, Becker Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy (EDMD), Myotonic Dystrophy, Fascioscapulohumeral Dystrophy (FSHD), Oculopharyngeal Muscular Dystrophy, Distal Muscular Dystrophy, cystic fibrosis, pulmonary fibrosis, muscle atrophy, cerebral palsy, an epithelial disorder, an epidermal disorder, a kidney disorder, a liver disorder, sarcopenia, cardiomyopathy, myopathy, cystic fibrosis, pulmonary fibrosis, cardiomyopathy (including hypertrophic, dilated, congenital, arrhythmogenic, restrictive, ischemic, or heart failure), acute lung injury, acute muscle injury, acute myocardial injury, radiation-induced injury, colon cancer, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia, autoimmune lung diseases, benign prostate hypertrophy, cerebral infarction, musculoskeletal fibrosis, post-surgical adhesions, liver cirrhosis, renal fibrotic disease, fibrotic vascular disease, neurofibromatosis, Alzheimer's disease, diabetic retinopathy, skin lesions, lymph node fibrosis associated with HIV, chronic obstructive pulmonary disease (COPD), inflammatory pulmonary fibrosis, rheumatoid arthritis; rheumatoid spondylitis; osteoarthritis; gout, other arthritic conditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic shock syndrome; myofacial pain syndrome (MPS); Shigellosis; asthma; adult respiratory distress syndrome; inflammatory bowel disease; Crohn's disease; psoriasis; eczema; ulcerative colitis; glomerular nephritis; scleroderma; chronic thyroiditis; Grave's disease; Ormond's disease; autoimmune gastritis; myasthenia gravis; autoimmune hemolytic anemia; autoimmune neutropenia; thrombocytopenia; pancreatic fibrosis; chronic active hepatitis including hepatic fibrosis; renal fibrosis, irritable bowel syndrome; pyresis; restenosis; cerebral malaria; stroke and ischemic injury; neural trauma; Huntington's disease; Parkinson's disease; allergies, including allergic rhinitis and allergic conjunctivitis; cachexia; Reiter's syndrome; acute synoviitis; muscle degeneration, bursitis; tendonitis; tenosynovitis; osteopetrosis; thrombosis; silicosis; pulmonary sarcosis; bone resorption diseases, such as osteoporosis or multiple myeloma-related bone disorders; cancer, including but not limited to metastatic breast carcinoma, colorectal carcinoma, malignant melanoma, gastric cancer, and non-small cell lung cancer; graft-versus-host reaction; and auto-immune diseases, such as multiple sclerosis, lupus and fibromyalgia; viral diseases such as Herpes Zoster, Herpes Simplex I or II, influenza virus, Severe Acute Respiratory Syndrome (SARS) and cytomegalovirus.
As used herein, “cardiomyopathy” refers to any disease or dysfunction of the myocardium (heart muscle) in which the heart is abnormally enlarged, thickened and/or stiffened. As a result, the heart muscle's ability to pump blood is usually weakened, often leading to congestive heart failure. The disease or disorder can be, for example, inflammatory, metabolic, toxic, infiltrative, fibrotic, hematological, genetic, or unknown in origin. Such cardiomyopathies may result from a lack of oxygen. Other diseases include those that result from myocardial injury which involves damage to the muscle or the myocardium in the wall of the heart as a result of disease or trauma. Myocardial injury can be attributed to many things such as, but not limited to, cardiomyopathy, myocardial infarction, or congenital heart disease. The cardiac disorder may be pediatric in origin. Cardiomyopathy includes, but is not limited to, cardiomyopathy (dilated, hypertrophic, restrictive, arrhythmogenic, genetic, idiopathic and unclassified cardiomyopathy), sporadic dilated cardiomyopathy, X-linked Dilated Cardiomyopathy (XLDC), acute and chronic heart failure, right heart failure, left heart failure, biventricular heart failure, congenital heart defects, myocardiac fibrosis, mitral valve stenosis, mitral valve insufficiency, aortic valve stenosis, aortic valve insufficiency, tricuspidal valve stenosis, tricuspidal valve insufficiency, pulmonal valve stenosis, pulmonal valve insufficiency, combined valve defects, myocarditis, acute myocarditis, chronic myocarditis, viral myocarditis, diastolic heart failure, systolic heart failure, diabetic heart failure and accumulation diseases.
In various embodiments of the disclosure it is contemplated that a second agent is administered with the agent that increases the activity of an annexin protein. Nonlimiting examples of the second agent are mitsugumin 53 (MG53), micro-dystrophin, a modulator of latent TGF-β binding protein 4 (LTBP4), a modulator of transforming growth factor β (TGF-β) activity, a modulator of androgen response, a modulator of an inflammatory response, a promoter of muscle growth, a chemotherapeutic agent, a modulator of fibrosis, and a combination thereof. Further, the methods disclosed herein can, in various embodiments, encompass one or more of such agents, or one or more of such agents in composition with any other active agent(s).
LTBP4 is located on human chromosome 19q13.1-q13.2, and is an extracellular matrix protein that binds and sequesters TGFβ. LTBP4 modifies murine muscular dystrophy through a polymorphism in the Ltbp4 gene. See U.S. Pat. No. 9,873,739, which is incorporated by reference herein in its entirety. There are two common variants of the Ltbp4 gene in mice. Most strains of mice, including the mdx mouse, have the Ltbp4 insertion allele (Ltbp4I/I). Insertion of 36 base pairs (12 amino acids) into the proline-rich region of LTBP4 encoded by Ltbp4I/I leads to milder disease. Deletion of 36 bp/12 aa in the proline-rich region is associated with more severe disease (Ltbp4D/D). It was found that the Ltbp4 genotype correlated strongly with two different aspects of muscular dystrophy pathology, i.e., membrane leakage and fibrosis, and these features define DMD pathology.
Modulators of LTBP4 are described in U.S. Pat. No. 9,873,739, which is incorporated by reference herein in its entirety.
Transforming Growth Factor-β (TGF-β) superfamily is a family of secreted proteins that is comprised of over 30 members including activins, nodals, bone morphogenic proteins (BMPs) and growth and differentiation factors (GDFs). Superfamily members are generally ubiquitously expressed and regulate numerous cellular processes including growth, development, and regeneration. Mutations in TGF-β superfamily members result in a multitude of diseases including autoimmune disease, cardiac disease, fibrosis and cancer.
TGF-β ligand family includes TGF-β1, TGF-β2, and TGF-β3. TGF-β is secreted into the extracellular matrix in an inactive form bound to latency associated peptide (LAP). Latent TGF-β proteins (LTBPs) binding the TGF-β/LAP complex provide yet another level of regulation. Extracellular proteases cleave LTBP/LAP/TGF-β releasing TGF-β. As a result, TGF-β is free to bind its receptors TGFBRI or TGFBRII. TGF-β/receptor binding, activates downstream canonical and non-canonical SMAD pathways, including activation of SMAD factors, leading to gene transcription. TGF-β signaling has emerged as a prominent mediator of the fibrotic response and disease progression in muscle disease and its expression is upregulated in dystrophy in both mouse and human. Blockade of TGF-β signaling in mice through expression of a dominant negative receptor (TGFBRII) expression, improved the dystrophic pathology, enhanced regeneration, and reduced muscle injury of 5-sarcoglycan-null mice, a mouse model of muscular dystrophy (Accornero, McNally et al Hum Mol Genet 2014). Additionally, antibody-mediated blockade of TGF-β signaling with a pan anti-TGF-β antibody, 1d11 monocloncal antibody, improved respiratory outcome measures in a mouse model of Duchenne muscular dystrophy (Nelson, Wentworth et al Am J Pathol 2011). Thus, therapeutic approaches against TGF-β signaling are contemplated herein to improve repair and delay disease progression.
Therapeutics contemplated as effective against TGF-β signaling include galunisertib (LY2157299 monohydrate), TEW-7917, monoclonal antibodies against TGF-β ligands (TGF-β 1, 2, 3 alone or pan 1,2,3), Fresolimemub (GC-1008), TGF-β peptide P144, LY2382770, small molecule, SB-525334, and GW788388.
Selective androgen receptor modulators (SARMs) are a class of androgen receptor ligands that activate androgenic signaling and exist in nonsteroidal and steroidal forms. Studies have shown that SARMs have the potential to increase both muscle and bone mass. Testosterone is one of the most well-known SARMs, which promotes skeletal muscle growth in healthy and diseased tissue. Testosterone and dihydrotestosterone (DHT) promote myocyte differentiation and upregulate follistatin, while also downregulates TGF-β signaling, resulting in muscle growth (Singh et al 2003, Singh et al 2009, Gupta et al 2008). It is conceivable that SARM-mediated inhibition of TGF-β protects against muscle injury and improves repair. SARMS may include, testosterone, estrogen, dihydrotestosterone, estradiol, include dihydronandrolone, nandrolone, nandrolone decanoate, Ostarine, Ligandrol, LGD-3303, andarine, cardarine, 7-alpha methyl, 19-nortestosterone aryl-propionamide, bicyclic hydantoin, quinolinones, tetrahydroquinoline analog, benizimidazole, imidazolopyrazole, indole, and pyrazoline derivatives, azasteroidal derivatives, and aniline, diaryl aniline, and bezoxazepinones derivatives.
A modulator of an inflammatory response includes the following agents. In one embodiment of the disclosure, the modulator of an inflammatory response is a beta2-adrenergic receptor agonist (e.g., albuterol). The term beta2-adrenergic receptor agonist is used herein to define a class of drugs which act on the 32-adrenergic receptor, thereby causing smooth muscle relaxation resulting in dilation of bronchial passages, vasodilation in muscle and liver, relaxation of uterine muscle and release of insulin. In one embodiment, the beta2-adrenergic receptor agonist for use according to the disclosure is albuterol, an immunosuppressant drug that is widely used in inhalant form for asthmatics. Albuterol is thought to slow disease progression by suppressing the infiltration of macrophages and other immune cells that contribute to inflammatory tissue loss. Albuterol also appears to have some anabolic effects and promotes the growth of muscle tissue. Albuterol may also suppress protein degradation (possibly via calpain inhibition).
In DMD, the loss of dystrophin leads to breaks in muscle cell membrane, and destabilizes neuronal nitric oxide synthase (nNOS), a protein that normally generates nitric oxide (NO). It is thought that at least part of the muscle degeneration observed in DMD patients may result from the reduced production of muscle membrane-associated neuronal nitric oxide synthase. This reduction may lead to impaired regulation of the vasoconstrictor response and eventual muscle damage.
In one embodiment, modulators of an inflammatory response suitable for use in compositions of the disclosure are Nuclear Factor Kappa-B (NF-κB) inhibitors. NF-κB is a major transcription factor modulating cellular immune, inflammatory and proliferative responses. NF-κB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. The activation of this factor in DMD contributes to diseases pathology. Thus, NF-κB plays an important role in the progression of muscular dystrophy and the IKK/NF-κB signaling pathway is a potential therapeutic target for the treatment of a TGFβ-related disease. Inhibitors of NF-κB (for example and without limitation, IRFI 042, a vitamin E analog) enhance muscle function, decrease serum creatine kinase (CK) level and muscle necrosis and enhance muscle regeneration. Edasalonexent is a small molecule inhibitor NF-κB. Edasalonexent administered orally as 100 mg/kg delayed muscle disease progression in Duchenne muscular dystrophy boys. Furthermore, specific inhibition of NF-κB-mediated signaling by IKK has similar benefits.
In a further embodiment, the modulator of an inflammatory response is a tumor necrosis factor alpha antagonist. TNF-α is one of the key cytokines that triggers and sustains the inflammation response. In one specific embodiment of the disclosure, the modulator of an inflammatory response is the TNF-α antagonist infliximab.
TNF-α antagonists for use according to the disclosure include, in addition to infliximab (Remicade™), a chimeric monoclonal antibody comprising murine VK and VH domains and human constant Fc domains. The drug blocks the action of TNF-α by binding to it and preventing it from signaling the receptors for TNF-α on the surface of cells. Another TNF-α antagonist for use according to the disclosure is adalimumab (Humira™) Adalimumab is a fully human monoclonal antibody. Another TNF-α antagonist for use according to the disclosure is etanercept (Enbrel™). Etanercept is a dimeric fusion protein comprising soluble human TNF receptor linked to an Fc portion of an IgG1. It is a large molecule that binds to TNF-α and thereby blocks its action. Etanercept mimics the inhibitory effects of naturally occurring soluble TNF receptors, but as a fusion protein it has a greatly extended half-life in the bloodstream and therefore a more profound and long-lasting inhibitory effect.
Another TNF-α antagonist for use according to the disclosure is pentoxifylline (Trental™), chemical name 1-(5-oxohexyl)-3,7-dimethylxanthine. The usual dosage in controlled-release tablet form is one tablet (400 mg) three times a day with meals.
Dosing: Remicade is administered by intravenous infusion, typically at 2-month intervals. The recommended dose is 3 mg/kg given as an intravenous infusion followed with additional similar doses at 2 and 6 weeks after the first infusion, then every 8 weeks thereafter. For patients who have an incomplete response, consideration may be given to adjusting the dose up to 10 mg/kg or treating as often as every 4 weeks. Humira is marketed in both preloaded 0.8 ml (40 mg) syringes and also in preloaded pen devices, both injected subcutaneously, typically by the patient at home. Etanercept can be administered at a dose of 25 mg (twice weekly) or 50 mg (once weekly).
In another embodiment of the disclosure, the modulator of an inflammatory response is cyclosporin. Cyclosporin A, the main form of the drug, is a cyclic nonribosomal peptide of 11 amino acids produced by the fungus Tolypocladium inflatum. Cyclosporin is thought to bind to the cytosolic protein cyclophilin (immunophilin) of immunocompetent lymphocytes (especially T-lymphocytes). This complex of cyclosporin and cyclophylin inhibits calcineurin, which under normal circumstances is responsible for activating the transcription of interleukin-2. It also inhibits lymphokine production and interleukin release and therefore leads to a reduced function of effector T-cells. It does not affect cytostatic activity. It has also an effect on mitochondria, preventing the mitochondrial PT pore from opening, thus inhibiting cytochrome c release (a potent apoptotic stimulation factor). Cyclosporin may be administered at a dose of 1-10 mg/kg/day.
In some embodiments of the disclosure, a therapeutically effective amount of a promoter of muscle growth is administered to a patient. Promoters of muscle growth contemplated by the disclosure include, but are not limited to, insulin-like growth factor-1 (IGF-1), Akt/protein kinase B, clenbuterol, creatine, decorin (see U.S. Patent Publication Number 20120058955), a steroid (for example and without limitation, a corticosteroid or a glucocorticoid steroid), testosterone and a myostatin antagonist.
Another class of promoters of muscle growth suitable for use in the combinations of the disclosure is myostatin antagonists. Myostatin, also known as growth/differentiation factor 8 (GDF-8) is a transforming growth factor-β (TGFβ) superfamily member involved in the regulation of skeletal muscle mass. Most members of the TGF-β-GDF family are widely expressed and are pleiotropic; however, myostatin is primarily expressed in skeletal muscle tissue where it negatively controls skeletal muscle growth. Myostatin is synthesized as an inactive preproprotein which is activated by proteolyic cleavage. The precursor protein is cleaved to produce an approximately 109-amino-acid COOH-terminal protein which, in the form of a homodimer of about 25 kDa, is the mature, active form. The mature dimer appears to circulate in the blood as an inactive latent complex bound to the propeptide. As used herein the term “myostatin antagonist” defines a class of agents that inhibits or blocks at least one activity of myostatin, or alternatively, blocks or reduces the expression of myostatin or its receptor (for example, by interference with the binding of myostatin to its receptor and/or blocking signal transduction resulting from the binding of myostatin to its receptor). Such agents therefore include agents which bind to myostatin itself or to its receptor.
Myostatin antagonists for use according to the disclosure include antibodies to GDF-8; antibodies to GDF-8 receptors; soluble GDF-8 receptors and fragments thereof (e.g., the ActRIIB fusion polypeptides as described in U.S. Patent Publication Number 2004/0223966, which is incorporated herein by reference in its entirety, including soluble ActRIIB receptors in which ActRIIB is joined to the Fc portion of an immunoglobulin); GDF-8 propeptide and modified forms thereof (e.g., as described in WO 2002/068650 or U.S. Pat. No. 7,202,210, including forms in which GDF-8 propeptide is joined to the Fc portion of an immunoglobulin and/or form in which GDF-8 is mutated at an aspartate (asp) residue, e.g., asp-99 in murine GDF-8 propeptide and asp-100 in human GDF-8 propeptide); a small molecule inhibitor of GDF-8; follistatin (e.g., as described in U.S. Pat. No. 6,004,937, incorporated herein by reference) or follistatin-domain-containing proteins (e.g., GASP-1 or other proteins as described in U.S. Pat. Nos. 7,192,717 and 7,572,763, each incorporated herein by reference); and modulators of metalloprotease activity that affect GDF-8 activation, as described in U.S. Patent Publication Number 2004/0138118, incorporated herein by reference.
Additional myostatin antagonists include myostatin antibodies which bind to and inhibit or neutralize myostatin (including the myostatin proprotein and/or mature protein, in monomeric or dimeric form). Myostatin antibodies are mammalian or non-mammalian derived antibodies, for example an IgNAR antibody derived from sharks, or humanized antibodies, or comprise a functional fragment derived from antibodies. Such antibodies are described, for example, in WO 2005/094446 and WO 2006/116269, the content of which is incorporated herein by reference. Myostatin antibodies also include those antibodies that bind to the myostatin proprotein and prevent cleavage into the mature active form. Additional antibody antagonists include the antibodies described in U.S. Pat. Nos. 6,096,506 and 6,468,535 (each of which is incorporated herein by reference). In some embodiments, the GDF-8 inhibitor is a monoclonal antibody or a fragment thereof that blocks GDF-8 binding to its receptor. Further embodiments include murine monoclonal antibody JA-16 (as described in U.S. Pat. No. 7,320,789 (ATCC Deposit No. PTA-4236); humanized derivatives thereof and fully human monoclonal anti-GDF-8 antibodies (e.g., Myo29, Myo28 and Myo22, ATCC Deposit Nos. PTA-4741, PTA-4740, and PTA-4739, respectively, or derivatives thereof) as described in U.S. Pat. No. 7,261,893 and incorporated herein by reference.
In still further embodiments, myostatin antagonists include soluble receptors which bind to myostatin and inhibit at least one activity thereof. The term “soluble receptor” herein includes truncated versions or fragments of the myostatin receptor that specifically bind myostatin thereby blocking or inhibiting myostatin signal transduction. Truncated versions of the myostatin receptor, for example, include the naturally occurring soluble domains, as well as variations produced by proteolysis of the N- or C-termini. The soluble domain includes all or part of the extracellular domain of the receptor, either alone or attached to additional peptides or other moieties. Because myostatin binds activin receptors (including the activin type IEB receptor (ActRHB) and activin type HA receptor (ActRHA)), activin receptors can form the basis of soluble receptor antagonists. Soluble receptor fusion proteins can also be used, including soluble receptor Fc (see U.S. Patent Publication Number 2004/0223966 and WO 2006/012627, both of which are incorporated herein by reference in their entireties).
Other myostatin antagonists based on the myostatin receptors are ALK-5 and/or ALK-7 inhibitors (see for example WO 2006/025988 and WO 2005/084699, each incorporated herein by reference). As a TGF-β cytokine, myostatin signals through a family of single transmembrane serine/threonine kinase receptors. These receptors can be divided in two classes, the type I or activin-like kinase (ALK) receptors and type II receptors. The ALK receptors are distinguished from the Type II receptors in that the ALK receptors (a) lack the serine/threonine-rich intracellular tail, (b) possess serine/threonine kinase domains that are highly homologous among Type I receptors, and (c) share a common sequence motif called the GS domain, consisting of a region rich in glycine and serine residues. The GS domain is at the amino terminal end of the intracellular kinase domain and is believed to be critical for activation by the Type II receptor. Several studies have shown that TGF-β signaling requires both the ALK (Type I) and Type II receptors. Specifically, the Type II receptor phosphorylates the GS domain of the Type 1 receptor for TGF3 ALK5, in the presence of TGFβ. The ALK5, in turn, phosphorylates the cytoplasmic proteins smad2 and smad3 at two carboxy terminal serines. Generally, it is believed that in many species, the Type II receptors regulate cell proliferation and the Type I receptors regulate matrix production. Various ALK5 receptor inhibitors have been described (see, for example, U.S. Pat. Nos. 6,465,493, 6,906,089, U.S. Patent Publication Numbers 2003/0166633, 2004/0063745 and 2004/0039198, the disclosures of which are incorporated herein by reference). Thus, the myostatin antagonists for use according to the disclosure may comprise the myostatin binding domain of an ALK5 and/or ALK7 receptor.
Other myostatin antagonists include soluble ligand antagonists that compete with myostatin for binding to myostatin receptors. The term “soluble ligand antagonist” herein refers to soluble peptides, polypeptides or peptidomimetics capable of non-productively binding the myostatin receptor(s) (e.g., the activin type HB receptor (ActRHA)) and thereby competitively blocking myostatin-receptor signal transduction. Soluble ligand antagonists include variants of myostatin, also referred to as “myostatin analogs” that have homology to, but not the activity of, myostatin. Such analogs include truncates (such as N- or C-terminal truncations, substitutions, deletions, and other alterations in the amino acid sequence, such as variants having non-amino acid substitutions).
Additional myostatin antagonists contemplated by the disclosure include inhibitory nucleic acids as described herein. These antagonists include antisense or sense polynucleotides comprising a single-stranded polynucleotide sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences. Thus, RNA interference (RNAi) produced by the introduction of specific small interfering RNA (siRNA), may also be used to inhibit or eliminate the activity of myostatin.
In specific embodiments, myostatin antagonists include, but are not limited to, follistatin, the myostatin prodomain, growth and differentiation factor 11 (GDF-11) prodomain, prodomain fusion proteins, antagonistic antibodies or antibody fragments that bind to myostatin, antagonistic antibodies or antibody fragments that bind to the activin type IEB receptor, soluble activin type IHB receptor, soluble activin type IEB receptor fusion proteins, soluble myostatin analogs (soluble ligands), polynucleotides, small molecules, peptidomimetics, and myostatin binding agents. Other antagonists include the peptide immunogens described in U.S. Pat. No. 6,369,201 and WO 2001/05820 (each of which is incorporated herein by reference) and myostatin multimers and immunoconjugates capable of eliciting an immune response and thereby blocking myostatin activity. Other antagonists include the protein inhibitors of myostatin described in WO 2002/085306 (incorporated herein by reference), which include the truncated Activin type II receptor, the myostatin pro-domain, and follistatin. Other myostatin inhibitors include those released into culture from cells overexpressing myostatin (see WO 2000/43781), dominant negative myostatin proteins (see WO 2001/53350) including the protein encoded by the Piedmontese allele, and mature myostatin peptides having a C-terminal truncation at a position either at or between amino acid positions 335 to 375. The small peptides described in U.S. Patent Publication Number 2004/0181033 (incorporated herein by reference) that comprise the amino acid sequence WMCPP, are also suitable for use in the compositions of the disclosure.
Chemotherapeutic agents contemplated for use include, without limitation, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p′-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.
A “modulator of fibrosis” as used herein is synonymous with antifibrotic agent. The term “antifibrotic agent” refers to a chemical compound that has antifibrotic activity (i.e., prevents or reduces fibrosis) in mammals. This takes into account the abnormal formation of fibrous connective tissue, which is typically comprised of collagen. These compounds may have different mechanisms of action, some reducing the formation of collagen or another protein, others enhancing the catabolism or removal of collagen in the affected area of the body. All such compounds having activity in the reduction of the presence of fibrotic tissue are included herein, without regard to the particular mechanism of action by which each such drug functions. Antifibrotic agents useful in the methods and compositions of the disclosure include those described in U.S. Pat. No. 5,720,950, incorporated herein by reference. Additional antifibrotic agents contemplated by the disclosure include, but are not limited to, Type II interferon receptor agonists (e.g., interferon-gamma); pirfenidone and pirfenidone analogs; anti-angiogenic agents, such as VEGF antagonists, VEGF receptor antagonists, bFGF antagonists, bFGF receptor antagonists, TGF3 antagonists, TGF3 receptor antagonists; anti-inflammatory agents, IL-1 antagonists, such as IL-1 Ra, angiotensin-converting-enzyme (ACE) inhibitors, angiotensin receptor blockers and aldosterone antagonists.
Gene correction approaches are contemplated by the disclosure to be used in conjunction with the methods and compositions as described herein. As used herein, “gene correction” approaches include, without limitation, technologies related to gene editing (i.e., CRISPR technology), exon skipping, and other technologies known in the art for modifying mRNA). Thus, in some embodiments, methods are provided in which an agent of the disclosure is used to increase the activity of an annexin protein in an individual suffering from Becker muscular dystrophy (BMD), Duchenne muscular dystrophy (DMD), all Limb Girdle muscular dystrophy (LGMD) type 1 subtypes, all LGMD type 2 subtypes, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), myotonic dystrophy, Fascioscapulohumeral dystrophy (FSHD), Oculopharyngeal muscular dystrophy, and Distal muscular dystrophy, wherein the patient will be, is concurrently being, or has previously been, administered a composition that results in correction of a gene involved in any one of the foregoing disorders. In further embodiments, methods are provided in which an agent of the disclosure is used to increase the activity of an annexin protein in an individual suffering from Becker muscular dystrophy (BMD), Duchenne muscular dystrophy (DMD), all Limb Girdle muscular dystrophy (LGMD) type 1 subtypes, all LGMD type 2 subtypes, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), myotonic dystrophy, Fascioscapulohumeral dystrophy (FSHD), Oculopharyngeal muscular dystrophy, and Distal muscular dystrophy, wherein the patient will be, is concurrently being, or has previously been, administered a viral-based or non-viral-based composition that results in correction of a gene involved in any one of the foregoing disorders.
Gene correction approaches are known in the art (see, e.g., U.S. Patent Application Publication No. 2016/0130608 and U.S. Pat. No. 9,499,817, respectively, each incorporated by reference herein in their entirety). Further discussion of such methods can be found in Echigoya et al., J Pers Med 8, 2018; Li et al., Trends Pharmacol Sci 39: 982-994, 2018; Min et al., Annu Rev Med, 2018; and Zhang et al., Physiol Rev 98: 1205-1240, 2018.
Any of the agents and/or additional agents described herein (or nucleic acids encoding any of the agents and/or additional agents described herein) also is provided in a composition. In this regard, the agent and/or additional agent is formulated with a physiologically-acceptable (i.e., pharmacologically acceptable) carrier, buffer, or diluent, as described further herein. Optionally, the protein/recombinant protein is in the form of a physiologically acceptable salt, which is encompassed by the disclosure. “Physiologically acceptable salts” means any salts that are pharmaceutically acceptable. Some examples of appropriate salts include acetate, trifluoroacetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, and oxalate. Accordingly, in some aspects the disclosure provides pharmaceutical compositions comprising one or more annexin proteins and a pharmaceutically acceptable carrier, buffer, and/or diluent. In any of the aspects or embodiments of the disclosure, one or more (or all) annexin proteins in a composition is a modified annexin protein. In any of the aspects or embodiments of the disclosure, one or more (or all) annexin proteins in a composition is a naturally-occurring mammalian annexin protein. In some embodiments, the modified annexin protein is expressed in a prokaryotic cell (for example and without limitation, an E. coli cell). In general, a modified protein is a protein that is altered relative to the version of the protein that normally exists in nature. In some embodiments, a modified protein is one in which at least one amino acid of the modified protein has an altered posttranslational modification relative to the naturally-occurring mammalian protein. By way of example, a naturally-occurring mammalian protein may comprise an amino acid that is phosphorylated while the same amino acid in the modified protein has either a different posttranslational modification or has no posttranslational modification. In some embodiments, the annexin protein is annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof), annexin A7 (SEQ ID NO: 9 or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 or SEQ ID NO: 18), or a combination thereof. In some embodiments, the annexin protein is annexin A6 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 45, or a combination thereof). In some embodiments, and as described herein, the pharmaceutical composition comprises a combination of annexin proteins wherein one or more of the annexin proteins is a modified annexin protein. In some embodiments, the pharmaceutical composition comprises a combination of annexin proteins and each annexin protein is a naturally-occurring mammalian annexin protein. Pharmaceutical compositions of the disclosure comprising one or more annexin proteins are formulated such that the one or more annexin proteins are present in the composition at a high level of purity. By “purity” it is meant that a protein (e.g., an annexin protein) used in a pharmaceutical composition is largely composed of the full-length protein (e.g., annexin protein) that was expressed and is largely free of truncated or degraded protein products. In various embodiments, the one or more annexin proteins that is/are present in a pharmaceutical composition is/are at least 90%, at least 95%, or at least 99% pure as measured by standard release assay including but not limited to one or more of SDS-PAGE, SEC-HPLC, and immunoblot analysis. A pharmaceutical composition of the disclosure is also relatively free of endotoxin. In various embodiments, a pharmaceutical composition of the disclosure has an endotoxin level that is or is less than about 10, is or is less than about 5, is or is less than about 1, is or is less than about 0.50000, is or is less than about 0.40000, is or is less than about 0.30000 endotoxin units per milligram (EU/mg) A280 annexin protein as determined by standard methods.
As disclosed herein, the disclosure provides compositions comprising one or more agents and/or additional agents that increase the activity of an annexin protein. In various embodiments, the annexin protein is annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 and/or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7 and/or SEQ ID NO: 8), annexin A7 (SEQ ID NO: 9 and/or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 and/or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 and/or SEQ ID NO: 16), annexin A13 (SEQ ID NO: 17 and/or SEQ ID NO: 18), or a combination thereof. In some embodiments, the composition increases the activity of annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 and/or SEQ ID NO: 3), and annexin A6 (SEQ ID NO: 7 and/or SEQ ID NO: 8). In further embodiments, the composition increases the activity of annexin A2 (SEQ ID NO: 2 and/or SEQ ID NO: 3) and annexin A6 (SEQ ID NO: 7 and/or SEQ ID NO: 8). In still further embodiments, the composition increases the activity of annexin A1 (SEQ ID NO: 1) and annexin A6 (SEQ ID NO: 7 and/or SEQ ID NO: 8).
The disclosure also contemplates, in various embodiments, compositions that increase the activity of annexin A1 (SEQ ID NO: 1), annexin A2 (SEQ ID NO: 2 and/or SEQ ID NO: 3), annexin A3 (SEQ ID NO: 4), annexin A4 (SEQ ID NO: 5), annexin A5 (SEQ ID NO: 6), annexin A6 (SEQ ID NO: 7 and/or SEQ ID NO: 8), annexin A7 (SEQ ID NO: 9 and/or SEQ ID NO: 10), annexin A8 (SEQ ID NO: 11 and/or SEQ ID NO: 12), annexin A9 (SEQ ID NO: 13), annexin A10 (SEQ ID NO: 14), annexin A11 (SEQ ID NO: 15 and/or SEQ ID NO: 16), and annexin A13 (SEQ ID NO: 17 and/or SEQ ID NO: 18) in any combination. Note that when more than one sequence identifier is used to identify an annexin protein herein (e.g., annexin A2 is identified herein by SEQ ID NO: 2 and/or SEQ ID NO: 3) it will be understood that the different sequence identifiers serve to identify isoforms of the particular annexin protein, and that the isoforms may be used interchangeably or in combination in methods and compositions of the disclosure.
sapiens] (SEQ ID NO: 2):
sapiens] (SEQ ID NO: 3):
sapiens] (SEQ ID NO: 5):
sapiens] (SEQ ID NO: 7):
sapiens] (SEQ ID NO: 8):
sapiens] (SEQ ID NO: 9):
sapiens] (SEQ ID NO: 10):
sapiens] (SEQ ID NO: 11):
sapiens] (SEQ ID NO: 12):
sapiens] (SEQ ID NO: 15):
sapiens] (SEQ ID NO: 16):
sapiens] (SEQ ID NO: 17):
sapiens] (SEQ ID NO: 18):
sapiens] (SEQ ID NO: 45):
The disclosure also contemplates corresponding polynucleotides that encode each of the foregoing annexin proteins. The following polynucleotides are contemplated for use according to the disclosure. Specifically, the following polynucleotides are messenger RNA (mRNA) sequences contemplated for use with a vector of the disclosure to increase activity of an annexin protein. As discussed above, when more than one sequence identifier is used to identify an mRNA sequence in relation to the same annexin species herein (e.g., mRNA sequences relating to annexin A2 are identified herein by SEQ ID NO: 20 and SEQ ID NO: 21) it will be understood that the different sequence identifiers serve to identify transcript variants that may be utilized with a vector of the disclosure to be translated into the particular annexin protein, and that the transcript variants may be used interchangeably or in combination in the methods and compositions of the disclosure.
In various aspects of the disclosure, use of the agent(s) and optional additional agent(s) as described herein provide one or more benefits related to specific therapeutic endpoints relative to a patient not receiving the agent(s) and/or additional agent(s).
Creatine kinase (CK) is a clinically validated serum biomarker of skeletal muscle, cardiac, kidney, and brain injury. Lactate dehydrogenase (LDH) is a clinically validated serum biomarker of skeletal muscle, cardiac, kidney, liver, lung, and brain injury. Creatine kinase and lactate dehydrogenase levels in serum are elevated with both acute and chronic tissue injury. In theoretical or verified conditions of comparable muscle mass levels, a reduction in creatine kinase and/or lactate dehydrogenase may be indicative of enhanced repair or protection against injury. Aspartate aminotransferase (AST) is yet another clinically validated serum biomarker of skeletal muscle, cardiac, kidney, liver, and brain injury. Additionally, increased serum troponin is indicative of cardiac injury, while elevated alanine transaminase (ALT) is a biomarker of liver injury. Reduction in AST, ALT, or troponin in the acute period following injury may indicate enhanced repair or protection against injury. Evan's blue due is a vital dye that binds serum albumin and is normally excluded from healthy, intact muscle. Membrane disruption due to acute or chronic injury promotes the influx of dye into the damaged cell. Evan's blue dye is commonly used to quantify cellular damage in experimental settings, measuring inherent dye fluorescence and/or through measuring radiolabeled-dye uptake. Reduction in dye uptake after acute injury or in models of chronic damage would indicate protection against injury and/or enhanced repair. Indocyanine green (ICG) is a near-infared dye that binds plasma proteins and is used clinically to evaluate blood flow and tissue damage (ischemia; necrosis) in organs including heart, liver, kidney, skin, vasculature, lung, muscle and eye. Improved blood flow and reduction in ischemic areas indicate protection from injury and/or improved repair.
It is contemplated that increasing membrane integrity and repair results in enhanced function measured through multiple modalities including plethysmography, echocardiography, muscle force, 6-min walk test. Additionally, histological benefits may be noted, including decreased necrosis, decreased inflammation, reduced fibrosis, reduced fatty infiltrate and reduced edema. These beneficial effects may also be visible through MR and PET imaging.
A particular administration regimen for a particular subject will depend, in part, upon the agent and optional additional agent used, the amount of the agent and optional additional agent administered, the route of administration, the particular ailment being treated, and the cause and extent of any side effects. The amount of agent and optional additional agent administered to a subject (e.g., a mammal, such as a human) is sufficient to effect the desired response. Dosage typically depends upon a variety of factors, including the particular agent and/or additional agent employed, the age and body weight of the subject, as well as the existence and severity of any disease or disorder in the subject. The size of the dose also will be determined by the route, timing, and frequency of administration. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain optimal therapeutic effect, and conventional range-finding techniques are known to those of ordinary skill in the art. Purely by way of illustration, in some embodiments, the method comprises administering an agent (e.g., a protein), e.g., from about 0.1 μg/kg up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 1 μg/kg up to about 75 mg/kg; or 5 μg/kg up to about 50 mg/kg; or 10 μg/kg up to about 20 mg/kg. In certain embodiments, the dose comprises about 0.5 mg/kg to about 20 mg/kg (e.g., about 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.3 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg) of agent and optional additional agent. In embodiments in which an agent and additional agent are administered, the above dosages are contemplated to represent the amount of each agent administered, or in further embodiments the dosage represents the total dosage administered. In some embodiments wherein a chronic condition is treated, it is envisioned that a subject will receive the agent and/or additional agent over a treatment course lasting weeks, months, or years, and may require one or more doses daily or weekly. In any of the aspects or embodiments of the disclosure, the amount of an annexin protein in a pharmaceutical composition is from about 0.1 μg/kg up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 1 μg/kg up to about 75 mg/kg; or 5 μg/kg up to about 50 mg/kg; or 10 μg/kg up to about 20 mg/kg. In some embodiments, the dose comprises about 0.5 mg/kg to about 20 mg/kg (e.g., about 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.3 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg) of annexin protein. Dosages are also contemplated for once daily, twice daily (BID) or three times daily (TID) dosing. A unit dose may be formulated in either capsule or tablet form. In other embodiments, the agent and optional additional agent is administered to treat an acute condition (e.g., acute muscle injury or acute myocardial injury) for a relatively short treatment period, e.g., one to 14 days.
Suitable methods of administering a physiologically-acceptable composition, such as a pharmaceutical composition comprising an agent (e.g., a recombinant protein) and optional additional agent described herein, are well known in the art. Although more than one route can be used to administer an agent and/or additional agent, a particular route can provide a more immediate and more effective avenue than another route. Depending on the circumstances, a pharmaceutical composition is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation. In some embodiments, a composition comprising an agent and/or additional agent is administered intravenously, intraarterially, or intraperitoneally to introduce an agent and optional additional agent into circulation. Non-intravenous administration also is appropriate, particularly with respect to low molecular weight therapeutics. In certain circumstances, it is desirable to deliver a pharmaceutical composition comprising the agent and/or additional agent orally, topically, sublingually, vaginally, rectally; through injection by intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intranasal, urethral, or enteral means; by sustained release systems; or by implantation devices. If desired, the agent and/or additional agent is administered regionally via intraarterial or intravenous administration to a region of interest, e.g., via the femoral artery for delivery to the leg. In one embodiment, the composition is administered via implantation of a membrane, sponge, or another appropriate material within or upon which the desired agent and optional additional agent has been absorbed or encapsulated. Where an implantation device is used, the device in one aspect is implanted into any suitable tissue, and delivery of the desired agent and/or additional agent is, in various embodiments, effected via diffusion, time-release bolus, or continuous administration. In other embodiments, the agent and optional additional agent is administered directly to exposed tissue during surgical procedures or treatment of injury, or is administered via transfusion of blood products. Therapeutic delivery approaches are well known to the skilled artisan, some of which are further described, for example, in U.S. Pat. No. 5,399,363.
In some embodiments facilitating administration, the agent and optional additional agent in one embodiment is formulated into a physiologically acceptable composition comprising a carrier (i.e., vehicle, adjuvant, buffer, or diluent). The particular carrier employed is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the agent and/or additional agent, by the route of administration, and by the requirement of compatibility with the recipient organism. Physiologically acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injectable use include, without limitation, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468). Injectable formulations are further described in, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia. Pa., Banker and Chalmers. eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986), incorporated herein by reference).
A pharmaceutical composition comprising an agent (e.g., a recombinant protein) and optional additional agent as provided herein is optionally placed within containers/kits, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents that may be necessary to reconstitute the pharmaceutical composition.
The disclosure thus includes administering to a subject one or more agent(s), in combination with one or more additional agent(s), each being administered according to a regimen suitable for that medicament. In some embodiments, the agent is a recombinant protein such as an annexin protein (e.g., annexin A6). Administration strategies include concurrent administration (i.e., substantially simultaneous administration) and non-concurrent administration (i.e., administration at different times, in any order, whether overlapping or not) of the agent and one or more additional agents(s). It will be appreciated that different components are optionally administered in the same or in separate compositions, and by the same or different routes of administration.
All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In addition, the entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. For example, where protein therapy is described, embodiments involving polynucleotide therapy (using polynucleotides/vectors that encode the protein) are specifically contemplated, and the reverse also is true. With respect to elements described as one or more members of a set, it should be understood that all combinations within the set are contemplated.
The following examples show results from experiments in which muscle membrane repair was monitored with real-time high-resolution imaging to visualize annexin cap formation and Ca2+ dynamics after laser-induced injury. It is demonstrated herein that overexpression of annexin proteins induced extracellular bleb formation at the site of membrane injury, which correlated with decreased intracellular Ca2+ accumulation. Pretreatment with recombinant annexin A6 prevented muscle injury in vitro and in vivo. Post-treatment with recombinant annexin A6 enhanced muscle repair in vivo. Overexpression of annexin A6 enhanced myofiber membrane repair, while overexpression of a dominant-negative annexin A6 mutant protein decreased membrane repair capacity. In addition, treatment with extracellular recombinant annexin A6 protein reduced laser-induced muscle damage of both wildtype and dystrophic myofibers. Moreover, administration of recombinant annexin A6 protected against toxin-induced muscle injury in vivo using multiple routes of administration. Furthermore, in dystrophic mice administration of recombinant annexin A6 reduced circulating levels of creatine kinase and lactate dehydrogenase, indicating enhanced membrane repair. The data provided herein show that annexin proteins are important agonists of membrane repair and that recombinant annexin A6 is a therapeutic target to treat conditions resulting from membrane fragility like muscle injury and muscular dystrophies.
A polymorphism in Anxa6, the gene encoding annexin A6, was previously identified in several commonly used experimental mouse strains that correlated with impaired muscle repair (Demonbreun et al., 2016a; Quattrocelli et al., 2017b; Swaggart et al., 2014). This polymorphism produced a truncated annexin A6 protein that interferes with sarcolemmal repair. Additional studies in mice have shown that loss of annexin A2 results in poor myofiber repair (Defour et al., 2017).
Annexin Ca2+ binding during muscle membrane repair was investigated and the kinetics of annexin A1, A2, and A6 repair cap formation after injury was assessed. Mutations in Ca2+-coordinating residues in annexin A1, A2, and A6 interfered with normal annexin repair cap formation. Annexin overexpression promoted the formation of external blebs at the site of membrane injury, which were released from the repair cap. Overexpression of annexin A6 resulted in the formation of larger blebs being released from the repair cap. These vesicles were enriched in Ca2+-binding protein GCaMP5G, which correlated with a reduction of intracellular Ca2+ fluorescence at the site of injury. Overexpression of annexin A6 reduced membrane injury, while mutation of E233, a critical Ca2+-coordinating residue in annexin A6 interfered with annexin repair complex formation, and correlated with decreased repair capacity. Local and systemic treatment with recombinant annexin A6 reduced muscle damage and promoted membrane repair in vivo. The data provided herein identify a new role for annexins in bleb release from muscle membrane lesions during membrane repair and identifies annexin A6 as a therapeutic target to enhance membrane repair capacity.
Animals. Wildtype mice from the 129T2/SvEmsJ background were bred and housed in a specific pathogen free facility on a 12-hour light/dark cycle and fed ad libitum in accordance with the Northwestern University's Institutional Animal Care and Use Committee regulations. 129T2/SvEmsJ (129T2) mice were purchased from the Jackson Laboratory (Ben Harbor, Me.; Stock #002065). mdx/hLTBP4 mice were generated as described in (Ceco et al., 2014; Quattrocelli et al., 2017b). sgcg-null mice were generated as described (Hack et al 1998). Two to three-month-old male and female were used for all experiments.
Plasmids. Plasmids encoding annexin A1, A2 and A6 with a carboxyl-terminal turboGFP tag were obtained from Origene (Rockville, Md.). Subcloning of annexin A1, A2, and A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix (Suwanee, Ga.). Site directed mutagenesis was performed by Mutagenix on annexin A1-GFP, A2-GFP and A6-GFP to create the Ca2+-binding mutants A1-D171A-GFP, A2-D161A-GFP, A6-D149A-GFP, and A6-E233A-GFP. Constructs were sequenced to verify mutagenesis. Plasmid DNA was isolated using the Qiagen endo-free Maxi prep kit (Qiagen #12362). The Ca2+ sensor GCaMP5G was purchased from (Addgene #31788).
Sequence comparison and protein schematics. Protein ribbon diagrams were generated using Swiss-PdbViewer using solved crystal structures of annexin A1 (1 MCX), A2 (2HYW), and annexin A6 (1AVC) available on www.rcsb.org. Clustal Omega from the European Bioinformatics Institute (EMBL-EBI) was used to align annexin mouse sequences from www.ncbi.nlm.nih.gov annexin A1 (NM_010730; SEQ ID NO: 37), A2 (NM_007585; SEQ ID NO: 38) and A6 (NM_013472: SEQ ID NO:39).
Electroporation, myofiber isolation, laser injury, cap and vesicle measurement. Flexor digitorum brevis (FDB) fibers were transfected with endo-free plasmid DNA by in vivo electroporation. Methods were described previously in (Demonbreun and McNally, 2015; Demonbreun et al., 2016b; DiFranco et al., 2009). Z-stack projections were acquired from consecutive acquisitions after the final time-lapse frame, approximately 4 minutes post damage, with a 0.125 μM step size between slices. Z-stack renderings were constructed in FIJI. Measurement of the cap area and feret diameter were conducted from a single slice near the middle of the z-stack using FIJI imaging tools. Fibers expressing similar levels of tagged or GCaMP5G protein were compared. GCaMP5G Ca2+ fluorescence was measured from the acquired timelapse images, using a standard rectangular ROI, placed inside the myofiber below the site of damage using FIJI. Fluorescence was expressed as F/F0. External vesicle number and GCaMP5G area were measured from endpoint z-stacks and max projection images using FIJI. Vesicles were considered external if they were found outside the sarcolemma assessed in brightfield and fluorescent channels. All measurements were acquired from myofibers isolated from at least n≥3 mice, n≥3 myofibers per mouse.
For recombinant myofibers studies, myofibers were isolated from mdx/hLTBP4 mice as described above. Myofibers were incubated in Ringer's media with or without 25 μg/ml recombinant annexin A6 (5186-A6-050, R&D systems). FM 4-64 (2.5 μm) was added to the myofibers just prior to imaging. Images were acquired and quantitated as described above. FM 4-64 area was measured using FIJI at imaging endpoint from a single slice near the middle of the z-stack. Z-stack step size (0.125 μm) was acquired from cap end to end.
Myofiber quality control was based on a number of characteristics including using adherent myofibers with intact sarcomere structure detected through brightfield imaging. Myofibers appeared devoid of tears or ruptures induced during the isolation protocol. The region of the myofiber selected for damage was linear and not located on a nucleus or neuromuscular junction. Additionally, fluorescence intensity within both the red and green channels suggested similar expression levels prior to damage.
Multiphoton laser injury and imaging. Fibers were subjected to laser-induced damage at room temperature using the Nikon A1R-MP multiphoton microscope. Imaging was performed using a 25×1.1 NA objective directed by the NIS-Elements AR imaging software. Green fluorescence protein (GFP) and FM 4-64 were excited using a 920 nm wavelength laser and emission wavelengths of 575 nm and 629 nm were collected respectively. To induce laser damage on isolated myofibers, a diffraction limited spot (diameter approximately 410 nm) was created on the lateral membrane of the myofiber using a 920 nm wavelength laser at 10-15% laser power for 1 second. Time lapse images were collected as follows: one image was collected prior to damage, one image upon damage, then every 8 seconds for 80 seconds (10 images) followed by every 30 seconds for 5 minutes (10 images). At the end of the time lapsed image series, z-stack images were collected at 250 nm intervals through the damaged site on the myofiber directed by the NIS-Elements AR imaging software.
Cardiotoxin Injury and analysis. Tibialis anterior muscle of wildtype mice were injected with 25 μg/ml recombinant annexin A6 (5186-A6-050, R&D systems) or Ringers in sedated mice (3% isoflurane, 0.8 l/min O2). Additionally, mice were injected intraperitoneally with Evans' blue dye at 5 μl/g body weight (E-2129; Sigma-Aldrich, St. Louis, Mo.) dissolved in phosphate-buffered saline at 10 mg/mL. For systemic administration, wildtype mice were injected with 1 mg/kg recombinant annexin A6 (5186-A6-050, R&D systems) or PBS diluted in EBD (5 μl/g body weight) into the retro-orbital cavity of sedated mice (3% isoflurane, 0.8 l/min O2). Cardiotoxin injury was performed injecting 20 μl of a 10 μM cardiotoxin (discontinued, Sigma-Aldrich) solution in PBS in tibialis muscles in sedated animals (3% isoflurane, 0.8 l/min O2) 2 hours post pretreatment. Cardiotoxin was released down the midline of the muscle to induce a homogenous area of injury at the center of the muscle. 3 hours post cardiotoxin injection muscle was harvested and frozen in liquid nitrogen.
Chronic Injury and biomarker analysis. For systemic administration, sgcg-null mice or wildtype mice were injected with 1 mg/kg recombinant annexin A6 (5186-A6-050, R&D systems) or PBS into the retro-orbital cavity of sedated mice (3% isoflurane, 0.8 l/min O2). Mice were injected once every 3 days for a period of 12 days. Serum was acquired as previously described (Demonbreun et al 2016a). Serum CK was analyzed for each mouse using the EnzyChrom Creatine Kinase Assay (catalog ECPK-100, BioAssay Systems) following the manufacturer's instructions. Lactate dehydrogenase was analyzed for each mouse using (LDH cytotoxicity kit MK401, Takara) following the manufacturer's instructions. Results were acquired with the Synergy HTX multi-mode plate reader (BioTeK).
Immunofluorescence microscopy. Sections (10 μm thick) from the center of frozen-embedded muscles were collected on the cryostat (chamber, −20° C.; sample, −15° C.; catalog number CM1950; Leica, Wetzlar, Germany) for immunostaining. Tissues were fixed with 4% paraformaldehyde for 10 minutes on ice. Block and permeabilization were with 0.1% Triton (catalog number X-100; Sigma-Aldrich), 10% fetal bovine serum, and PBS for 60 minutes. For dystrophin detection, anti-dystrophin (ab15277; Abcam, Cambridge, Mass.) was used at a dilution of 1:100 overnight at 4° C. Sections were PBS rinsed, incubated with secondary antibody goat anti rabbit (A11008, Invitrogen) for 1 hour, PBS rinsed, and mounted in vectashield with DAPI (H-1200, Vector Laboratories). Imaging was performed using a Zeiss Axio Observer A1 microscope (Zeiss, Oberkochen, Germany), using a 10× objective. ZEN software (Zeiss, Jena, Germany) was used for acquiring images. Fluorescence quantitation and muscle area were performed using FIJI (NIH). Surface plots were created in FIJI (NIH). Fluorescence volume was quantified using Imaris Software v9.1.2.
Calcium kinetics. FDB muscle was electroporated and isolated as described above. Myofibers were damaged in Ringers solution with Ca2+ concentrations of 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.175 mM, 0.1 mM, 0.050 mM, and 0 mM. EDTA was added as a Ca2+ chelating agent in only in the 0 mM Ca2+ Ringers. Myofibers were isolated directly into 2 mM, 1 mM and 0.5 mM Ringers for those experiments respectively. For experiments using less than 0.5 mM Ca2+ myofibers were isolated in 0.5 mM Ca2+ Ringers and then diluted with 0 mM EDTA-free Ca2+ Ringers. For 0 mM experiments, myofibers were isolated in 0.5 mM Ca2+ Ringers and then replaced with 0 mM Ca2++EDTA Ringers just prior to imaging. Co-electroporation of wildtype annexin+wildtype annexin constructs was performed in one mouse foot, while the contralateral foot was co-electroporated with wildtype annexin+mutant annexin. All measurements were acquired from myofibers isolated from at least n≥2 mice, n≥3 myofibers per mouse at each Ca2+ concentration. Area-Ca2+ curves were fitted with a Hill Curve at Ca2+ concentrations ranging from 0-2 mM. Kinetic parameters were calculated using Prism Graphpad.
Single cell Ca2+ and shortening measurements. Isolated FDB fibers were plated on laminin coated glass-bottomed 35 mm dishes for one hour and then cultured overnight in DMEM with 10% FBS and 1% penicillin/streptomycin at 37° C. in a 10% CO2 incubator. One hour prior to data acquisition, the medium was removed and cells were incubated in Tyrode buffer (119 mM NaCl, 5 mM KCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2) with 10 μM Indo-1 AM (TefLabs) for 1 hour at 37° C. in a 10% CO2 incubator. Dishes were then filled with Tyrode buffer, mounted on a custom stage and platinum pacing electrodes were inserted into the dish. Stimulation was elicited using a 701C high-powered stimulator controlled by the 950A software (Aurora Scientific). Stimulation was performed at 40 and 80 Hz, 5 ms pulse width, 100 ms duration. Ratiometric Ca2+ signals were collected with two photomultiplier tubes and a FluoroDaq controller. Video sarcomere length was recorded with a high-speed camera and fast Fourier transform using the Aurora Scientific 900B-VSL system (Aurora, Ontario). Ten transients were collected over 20 seconds and averaged together per cell per frequency.
Statistical analysis. Statistical analyses were performed with Prism (Graphpad, La Jolla, Calif.). Comparisons relied on ANOVA ((1way ANOVA for 1 variable, 2way ANOVA for two variables (typically area and Ca2+ concentration)). Otherwise, unpaired t-tests were performed. Error bars represent +/−standard error of the mean (SEM).
Activation of muscle membrane repair requires the presence of external Ca2+ (Bansal et al., 2003). It was previously shown that annexin proteins aggregate into repair caps at the site of injury bordered by annexin-free zone within the cytoplasm under the repair cap (Demonbreun et al., 2016a; Demonbreun et al., 2016b; Quattrocelli et al., 2017a; Swaggart et al., 2014). To visualize Ca2+ dynamics at the site of injury in real-time, an in vivo fluorescent Ca2+ indicator protein, GCaMP5G, was utilized. GCaMP5G is a fusion protein composed of green fluorescent protein (GFP), the calcium-binding protein calmodulin, and the calmodulin M13 binding peptide. GCaMP5G has minimal fluorescence when not bound to Ca2+, and Ca2+ binding results in a conformational change within the protein increasing the fluorescence intensity of GFP (Akerboom et al., 2012). Wildtype flexor digitorum brevis (FDB) muscle was electroporated with the GCaMP5G plasmid and then injured the plasma membrane using laser ablation (Demonbreun and McNally, 2015; Demonbreun et al., 2016b). Within two seconds of membrane injury, GCaMP5G fluorescence accumulated in the cytoplasm at the site of injury. GCaMP5G fluorescence intensity progressively increased through 90 seconds of imaging (
Annexin proteins are Ca2+-dependent phospholipid and actin binding proteins that contain four annexin repeat domains or eight in the case of annexin A6 (
Membrane repair assays in Lytechinus pictus and Xenopus oocytes suggested that membranous structures merge and erupt at the site of membrane repair (Bi et al., 1995; Davenport et al., 2016). Additionally, extracellular recombinant annexins promoted membrane folding and bleb formation of artificial membrane patches at sites of membrane imperfection in a Ca2+ dependent manner (Boye et al., 2018; Boye et al., 2017). It was next investigated whether similar findings could be observed at the site of muscle membrane injury, which are sites of membrane imperfection, in live skeletal myofibers. GCaMP5G was expressed alone or in combination with annexin A1, A2 or A6 in skeletal myofibers. Overexpression of annexins was found to promote the formation of extracellular blebs emanating from annexin repair caps at the membrane lesion (
Time lapse imaging of the Ca2+ indicator GCaMP5G after laser injury suggested intracellular Ca2+ was decreasing concomitant with extracellular bleb formation suggesting that these blebs serve to reduce intracellular Ca2+ accumulation and/or excretion of cytoplasmic protein content emanating from the site of injury. The annexin-induced reduction in intracellular Ca2+ fluorescence was seen for all three annexins A1, A2 and A6, but was most evident for annexin A2 and A6 (
Next, the Ca2+ handling and contractile properties of isolated myofibers overexpressing annexin A6 compared to controls was evaluated. Isolated myofibers expressing annexin A6 were loaded with the ratiometric Ca2+ indicator dye Indo-1, and no differences in Ca2+ cycling at 40 or 80 Hz stimulation frequencies between annexin A6 or control fibers (
Mutation of annexin A1 D171 and A2D161 were previously shown to inhibit annexin membrane translocation in HEK cells (Jost et al., 1992; McNeil et al., 2006). It was queried whether these mutations would inhibit translocation and formation of the macromolecular annexin repair cap formed after muscle membrane injury. Alignment of annexins A1, A2 and A6 protein sequences identified conserved residues within the consensus sequence of type II Ca2+ binding sites across all three annexin proteins (
Ca2+-binding of both annexin A1 and A2 was also required for repair cap formation. A1 D171A and A2D161A mutant cap size was reduced compared to wildtype annexin A1 and A2 controls, respectively. Expression of mutant annexin A1 D171A and A2D161A was sufficient to significantly reduce the repair cap diameter (DMAX) of the respective co-expressed wildtype annexin protein (
To determine the effect of dominant negative annexin A6 on the assembly of annexins A1, A2, and A6 at the repair cap and membrane repair capacity, laser injury was similarly performed on isolated myofibers in the presence of FM 4-64. FM 4-64 is a membrane impermeable dye that is non-fluorescent in aqueous solution and increases fluorescence intensity as it binds membrane phospholipids exposed during injury, and is commonly used as a marker of membrane injury (Bansal et al., 2003; Cai et al., 2009; Demonbreun and McNally, 2015; Yeung et al., 2009; Zweifach, 2000). Myofibers expressing annexin A6E233A-GFP had increased FM 4-64 fluorescence area after laser injury compared to control myofibers expressing wildtype annexin A6-GFP (
Since annexin A6 facilitates the formation of the macromolecular repair cap complex and was the most efficient at forming large, Ca2+-filled blebs at the site of membrane injury, whether overexpression of annexin A6 would reduce membrane injury in wildtype myofibers was assessed. Wildtype myofibers were electroporated with annexin A6-GFP or mock electroporated and then laser damaged in the presence of FM 4-64, to mark the injury area. Wildtype myofibers overexpressing annexin A6 had decreased FM 4-64 dye uptake after laser-induced membrane injury compared to control myofibers (
Next, it was tested whether extracellular recombinant annexin A6 could also protect against membrane injury in wildtype myofibers. Wildtype myofibers were isolated and incubated with recombinant annexin A6 (rANXA6) or vehicle control. Laser injury was conducted in the presence of FM 4-64. Pretreatment with extracellular recombinant annexin A6 reduced FM 4-64 fluorescence area compared to vehicle control treated myofibers (
Muscular dystrophy is a progressive muscle wasting disease, due to loss-of-function mutations in critical cytoskeletal or membrane associated proteins, that results in fragile membranes. mdx mice lack the integral membrane protein dystrophin and are a model of Duchenne muscular dystrophy (DMD) (Hoffman et al., 1987; Petrof et al., 1993). mdx mice expressing human Latent TGFβ binding protein 4 (mdx/hLTBP4) have a more severe form of muscular dystrophy, similar to what is seen in humans with DMD (Ceco et al., 2014; Flanigan et al., 2013; Quattrocelli et al., 2017b). Since recombinant annexin A6 protected against laser-induced membrane injury in wildtype muscle, it was next assessed whether exposure to recombinant annexin A6 would protect against membrane injury in the context of chronic muscle disease. anti-HIS (green) immunostaining revealed the presence of recombinant annexin A6-HIS at the sarcolemma of mdx/hLTBP4 dystrophic muscle after systemic injection, while vehicle control muscle lacked anti-HIS staining (
It was next assessed whether recombinant annexin A6 could protect against muscle injury in vivo. Recombinant annexin A6 or vehicle control was injected intramuscularly into the tibialis anterior (TA) muscles of wildtype mice. Mice were also injected intraperitoneally with Evan's blue dye, a vital tracer that is excluded by intact healthy myofibers but is readily taken up in injured permeable myofibers (Jennische and Hansson, 1986). Two hours post injection of recombinant annexin A6, the TA muscle was injured with cardiotoxin. Muscle was harvested 3 hours post-cardiotoxin injury and evaluated for Evan's blue dye uptake (
Although intramuscular injection of annexin A6 was effective at reducing injury, this route of application is not optimal for large muscle groups, internal tissues, or treatment of chronic diseases. Therefore, the efficacy of recombinant annexin A6 administered via systemic retro-orbital (RO) injection was examined. Recombinant annexin A6 or control solution was injected 2 hours prior to cardiotoxin-induced tibialis anterior muscle injury (pretreatment). Alternatively, recombinant annexin A6 was administered immediately after tibialis anterior (TA) muscles were injured with cardiotoxin (post-treatment). Additionally, Evan's blue dye, was injected prior to injury. Muscle was harvested 3 hours post cardiotoxin injury and evaluated for dye uptake (
Since recombinant annexin A6 protected against acute membrane injury in wildtype muscle in vivo, it was next assessed whether exposure to recombinant annexin A6 would protect against membrane injury in the context of chronic muscle disease. Mutations in the gene γ-sarcoglycan (SGCG) cause Limb-Girdle muscular dystrophy 2C (LGMD2C) in both mice and humans. To determine the if recombinant annexin A6 could facilitate repair in a chronic injury setting, sgcg-null mice were injected once every 3 days for 12 days with recombinant protein or control solution and then serum biomarkers of muscle damage, creatine kinase (CK) and lactate dehydrogenase (LDH), were quantified (
Annexins promote bleb formation at the site of membrane injury. It was found that increased expression of annexins in muscle fibers decreased injury-associated Ca2+ fluorescence accumulation within myofibers. This reduction correlated with extracellular bleb formation arising at the site of annexin repair caps. We found that annexin A2 and A6 induced the formation of membranous blebs containing the Ca2+-binding protein GCaMP5G emanating from the repair cap. Furthermore, overexpression of annexins A1, A2, and A6 each reduced endpoint Ca2+ fluorescence accumulation within the myofiber after injury. Annexin A6 overexpression resulted in the most sustained effect on reducing injury-associated Ca2+ accumulation inducing the formation of large GCaMP5G-containing blebs. A model is contemplated in which annexin A6 facilitates Ca2+ and protein excretion into blebs whose formation was further induced by annexin A1 and annexin A2. In artificial membrane patches, the presence of annexin A1 or annexin A2 induced bleb formation at sites of membrane imperfection (Boye et al., 2018). In contrast, the presence of annexin A6 induced contraction of artificial membrane into large folds, in a Ca2+-dependent manner (Boye et al., 2018). The difference between annexin A6 inducing blebs in live myofibers or folds in artificial membrane is likely do to the presence of endogenously expressed annexin A1 and A2 in isolated myofibers compared with exposure to single recombinant annexin protein in the artificial membrane studies. Without being bound by theory, it is hypothesized that within the macromolecular repair complex, annexins actively participate in bleb formation which acts to remove large membrane lesions facilitating wound closure, excision of damaged membrane, and reduction of Ca2+ at the injury site.
Similar to the data described herein, others have shown that in damaged Xenopus oocytes, GCaMP5G fluorescence quickly localized in a ring around the site of membrane disruption, fading by approximately 5 minutes post injury, as healing progressed (Davenport et al., 2016). Overexpression of annexin A1-GFP in injured Xenopus oocytes resulted in annexin A1 positive blebs originating from the site of damage (Davenport et al., 2016). However, the effect of annexin overexpression on GCaMP5G fluorescence was not assessed. These data combined suggest that bleb formation, as a mechanism of membrane repair, is conserved across species and tissue types, and is facilitated by the presence of annexin proteins.
Annexin A6 protects against muscle membrane injury and enhances membrane repair. As shown herein, annexin proteins, including annexin A1, A2, and A6, localize to the site of membrane injury facilitating membrane repair cap and bleb formation. Mutation of annexin A6 abrogated repair cap formation, decreasing repair capacity, resulting in increased dye uptake. On the other hand, pretreatment with recombinant annexin A6 reduced dye uptake after laser-induced muscle injury and after toxin-induced muscle injury in vivo. This data, however, does not distinguish between annexin A6 enhancing membrane repair, reducing membrane injury, or a combination of both mechanisms. As a therapeutic tool, enhancing the cells' ability to repair and/or reduce injury through stabilizing the cell membrane are both beneficial avenues that can lead to improved cell survival. Previous studies have shown that annexin A6 is upregulated in muscle from models of chronic muscular dystrophy (Demonbreun et al., 2016a; Demonbreun et al., 2014; Swaggart et al., 2014). Additional proteomic approaches in the mdx mouse model have shown that annexin A1, A2, and A6 are enriched in mdx muscle membrane, again, suggesting a role for annexins at the membrane of injured cells (Murphy et al., 2018). Annexins bind membrane phospholipids, including phosphatidylserine, which is exposed during membrane disruption. Phosphatidylserine rearrangement upon injury provides a likely binding target for extracellular annexins to facilitate membrane folding, blebbing, and rolling at sites of membrane damage and imperfection (Boye et al., 2018). Based on the data herein, upregulation of annexin A6 is a compensatory mechanism to facilitate excision of defective membrane in fibers undergoing chronic damage. It is further contemplated that recombinant annexin A6 can facilitate membrane repair and reduce the susceptibility to injury long-term in chronic models of disease and in tissues beyond skeletal muscle.
Combinatorial approaches to improve membrane repair. As shown herein, recombinant annexin A6 protected normal and dystrophic muscle from laser-induced membrane injury. In addition, both intramuscular and systemic administration of recombinant annexin A6 protected against toxin-induced muscle membrane injury in vivo. Intriguingly, glucocorticoid administration increased annexin expression in muscle and this correlated with enhanced muscle repair in multiple mouse models of muscular dystrophy including mdx (DMD), dysferlin-null (LGMD2B), and γ-sarcoglycan-null (LGMD2C) mice (Quattrocelli et al., 2017a; Quattrocelli et al., 2017c). Importantly, glucocorticoid treatment also increased the expression of mitsugumin 53 (also known as MG53 or Trim 72), a repair protein that localizes to the site of membrane injury and is considered a “molecular band-aid” improving cellular wound healing. Similar to the annexins, MG53 is upregulated in chronic muscle injury and enhances repair in dystrophic muscles, as well as other tissues like heart, lung, kidney (Waddell et al., 2011). (Duann et al., 2015; He et al., 2012; Jia et al., 2014; Liu et al., 2015; Weisleder et al., 2012). MG53 is a component of the annexin-mediated repair complex, localizing juxtaposed to the annexin repair cap (Demonbreun et al., 2016b). It is contemplated that co-administration of recombinant annexin A6 with glucocorticoids and/or MG53 will further strengthen clinical relevance of these therapeutics for conditions resulting from membrane lesions.
In summary, the data provided herein demonstrate that annexins promote the formation of blebs released at the site of muscle membrane insult, with annexin A6 being the most effective at facilitating this process. Furthermore, recombinant annexin A6 protects against membrane injury of normal and dystrophic muscle. These data identify annexin A6 as a therapeutic target that enhances membrane repair capacity in healthy and diseased muscle.
This example details the results of additional and updated experiments that were performed.
Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated Ca2+-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, annexin cap formation was visualized during injury. Annexin cap size was found to be positively correlated with increasing Ca2+ concentrations. It was also found that annexin overexpression promoted external blebs enriched in Ca2+ fluorescence and correlated with a reduction of intracellular Ca2+ fluorescence at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identified annexins as mediators of membrane-associated Ca2+ release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity.
Annexin A6 forms a repair cap at the site of muscle membrane disruption. The data herein demonstrated that adding exogenous recombinant annexin A6 (rANXA6) promotes membrane resealing and recovery from injury.
Plasma membrane repair occurs after membrane disruption and is a highly conserved process. The active process required for resealing membrane disruptions is thought to rely on Ca2+-dependent vesicle fusion and local cytoskeletal remodeling (McNeil P L, and Khakee R. Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage. Am J Pathol. 1992; 140(5):1097-109; McNeil P L, and Kirchhausen T. An emergency response team for membrane repair. Nat Rev Mol Cell Biol. 2005; 6(6):499-505). Other models suggest that membrane repair is mediated through the fusion of lysosomal vesicles, lateral diffusion of membrane to the site of injury, and the extrusion of membranous blebs (Rodriguez A, Webster P, Ortego J, and Andrews N W. Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. J Cell Biol. 1997; 137(1):93-104; Reddy A, Caler E V, and Andrews N W. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell. 2001; 106(2):157-69; Demonbreun A R, Quattrocelli M, Barefield D Y, Allen M V, Swanson K E, and McNally E M. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016; 213(6):705-18; McDade J R, Archambeau A, and Michele D E. Rapid actin-cytoskeleton-dependent recruitment of plasma membrane-derived dysferlin at wounds is critical for muscle membrane repair. FASEB J. 2014; 28(8):3660-70; Babiychuk E B, Monastyrskaya K, Potez S, and Draeger A. Blebbing confers resistance against cell lysis. Cell Death Differ. 2011; 18(1):80-9). These models are not mutually exclusive and may depend on the type and extent of damage. Skeletal muscle is highly dependent on plasma membrane repair as mutation in genes encoding repair proteins lead to muscle disease (Bansal D, Miyake K, Vogel S S, Groh S, Chen C C, Williamson R, et al. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature. 2003; 423(6936):168-72; Bashir R, Britton S, Strachan T, Keers S, Vafiadaki E, Lako M, et al. A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B. Nat Genet. 1998; 20(1):37-42; Demonbreun A R, Swanson K E, Rossi A E, Deveaux H K, Earley J U, Allen M V, et al. Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle. PLoS One. 2015; 10(9):e0136679; Demonbreun A R, and McNally E M. Plasma Membrane Repair in Health and Disease. Curr Top Membr. 2016; 77:67-96; Defour A, Medikayala S, Van der Meulen J H, Hogarth M W, Holdreith N, Malatras A, et al. Annexin A2 links poor myofiber repair with inflammation and adipogenic replacement of the injured muscle. Human molecular genetics. 2017; 26(11):1979-91; Cai C, Masumiya H, Weisleder N, Matsuda N, Nishi M, Hwang M, et al. MG53 nucleates assembly of cell membrane repair machinery. Nat Cell Biol. 2009; 11(1):56-64).
The annexins are a family of Ca2+-binding proteins that regulate lipid binding, cytoskeletal reorganization, and membrane folding, steps necessary for membrane repair (Jimenez A J, and Perez F. Plasma membrane repair: the adaptable cell life-insurance. Curr Opin Cell Biol. 2017; 47:99-107; Lauritzen S P, Boye T L, and Nylandsted J. Annexins are instrumental for efficient plasma membrane repair in cancer cells. Semin Cell Dev Biol. 2015; 45:32-8; Bizzarro V, Petrella A, and Parente L. Annexin A1: novel roles in skeletal muscle biology. J Cell Physiol. 2012; 227(8):3007-15; Grewal T, Hoque M, Conway J R W, Reverter M, Wahba M, Beevi S S, et al. Annexin A6-A multifunctional scaffold in cell motility. Cell Adh Migr. 2017; 11(3):288-304; Boye T L, Jeppesen J C, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018; 8(1):10309; Boye T L, Maeda K, Pezeshkian W, Sonder S L, Haeger S C, Gerke V, et al. Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair. Nat Commun. 2017; 8(1):1623). Individual annexin repeat domains coordinate Ca2+ binding with unique annexin-specific type II or type Ill binding sites. Differential Ca2+ affinity of the type II and type Ill binding sites provides each annexin a unique ability to respond to a range of intracellular Ca2+ levels and phospholipid binding (Blackwood R A, and Ernst J D. Characterization of Ca2(+)-dependent phospholipid binding, vesicle aggregation and membrane fusion by annexins. The Biochemical journal. 1990; 266(1):195-200). Annexins have the ability to self- and hetero-oligomerize (Zaks W J, and Creutz C E. Ca(2+)-dependent annexin self-association on membrane surfaces. Biochemistry. 1991; 30(40):9607-15). Typical annexins like A1 and A2 contain one annexin core composed of four annexin repeat domains. In contrast, annexin A6 contains two annexin cores and thus eight annexin repeat domains (Benz J, Bergner A, Hofmann A, Demange P, Gottig P, Liemann S, et al. The structure of recombinant human annexin VI in crystals and membrane-bound. J Mol Biol. 1996; 260(5):638-43). Annexin A6's duplicated structure makes it possible for the amino- and carboxyl-terminal annexin core domains to bind one or two distinct membranes making annexin A6 a prime target for facilitating membrane coalescence and folding required during membrane repair (Boye T L, Jeppesen J C, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018; 8(1):10309; Boye T L, Maeda K, Pezeshkian W, Sonder S L, Haeger S C, Gerke V, et al. Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair. Nat Commun. 2017; 8(1):1623; Buzhynskyy N, Golczak M, Lai-Kee-Him J, Lambert O, Tessier B, Gounou C, et al. Annexin-A6 presents two modes of association with phospholipid membranes. A combined QCM-D, AFM and cryo-TEM study. Journal of structural biology. 2009; 168(1):107-16).
Annexins have a high affinity for phosphatidylserine, phosphatidylinositol, and cholesterol, which are highly enriched in the sarcolemma (Gerke V, Creutz C E, and Moss S E. Annexins: linking Ca2+ signalling to membrane dynamics. Nat Rev Mol Cell Biol. 2005; 6(6):449-61; Fiehn W, Peter J B, Mead J F, and Gan-Elepano M. Lipids and fatty acids of sarcolemma, sarcoplasmic reticulum, and mitochondria from rat skeletal muscle. The Journal of biological chemistry. 1971; 246(18):5617-20). Multiple annexins, including annexins A1, A2, and A6, have been implicated in membrane repair in skeletal muscle, as well as Xenopus oocytes, human trophoblasts, and HeLa cancer cells, suggesting a conserved mechanism (Demonbreun A R, Quattrocelli M, Barefield D Y, Allen M V, Swanson K E, and McNally E M. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016; 213(6):705-18; Babbin B A, Laukoetter M G, Nava P, Koch S, Lee W Y, Capaldo C T, et al. Annexin A1 regulates intestinal mucosal injury, inflammation, and repair. J Immunol. 2008; 181(7):5035-44; Lennon N J, Kho A, Bacskai B J, Perlmutter S L, Hyman B T, and Brown R H, Jr. Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing. The Journal of biological chemistry. 2003; 278(50):50466-73; McNeil A K, Rescher U, Gerke V, and McNeil P L. Requirement for annexin A1 in plasma membrane repair. The Journal of biological chemistry. 2006; 281(46):35202-7; Roostalu U, and Strahle U. In vivo imaging of molecular interactions at damaged sarcolemma. Dev Cell. 2012; 22(3):515-29; Davenport N R, Sonnemann K J, Eliceiri K W, and Bement W M. Membrane dynamics during cellular wound repair. Mol Biol Cell. 2016; 27(14):2272-85; Carmeille R, Degrelle S A, Plawinski L, Bouvet F, Gounou C, Evain-Brion D, et al. Annexin-A5 promotes membrane resealing in human trophoblasts. Biochimica et biophysica acta. 2015; 1853(9):2033-44; Bement W M, Mandato C A, and Kirsch M N. Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes. Curr Biol. 1999; 9(11):579-87). Annexins are recruited to the injured membrane in a sequential manner forming a macromolecular repair complex at the membrane lesion referred to as a repair cap (Demonbreun A R, Quattrocelli M, Barefield D Y, Allen M V, Swanson K E, and McNally E M. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016; 213(6):705-18; Boye T L, Maeda K, Pezeshkian W, Sonder S L, Haeger S C, Gerke V, et al. Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair. Nat Commun. 2017; 8(1):1623; Roostalu U, and Strahle U. In vivo imaging of molecular interactions at damaged sarcolemma. Dev Cell. 2012; 22(3):515-29). A polymorphism in Anxa6, the gene encoding annexin A6, was previously identified in several commonly used experimental mouse strains that correlated with impaired muscle repair (Demonbreun A R, Allen M V, Warner J L, Barefield D Y, Krishnan S, Swanson K E, et al. Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol. 2016; 186(6):1610-22; Quattrocelli M, Capote J, Ohiri J C, Warner J L, Vo A H, Earley J U, et al. Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury. PLoS Genet. 2017; 13(10):e1007070; Swaggart K A, Demonbreun A R, Vo A H, Swanson K E, Kim E Y, Fahrenbach J P, et al. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair. Proceedings of the National Academy of Sciences of the United States of America. 2014; 111(16):6004-9). This polymorphism produces a truncated annexin A6 protein that acts in a dominant-negative manner to reduce repair cap formation and interferes with sarcolemmal repair. Additional studies in mice have shown that loss of annexin A2 results in poor myofiber repair (Defour A, Medikayala S, Van der Meulen J H, Hogarth M W, Holdreith N, Malatras A, et al. Annexin A2 links poor myofiber repair with inflammation and adipogenic replacement of the injured muscle. Human molecular genetics. 2017; 26(11):1979-91). These data suggest that there is a coordinated recruitment of annexin proteins to the repair cap facilitated by dynamic protein-protein interactions.
Herein, the kinetics of annexin A1, A2, and A6 were assessed in repair cap formation after membrane injury at multiple Ca2+ concentrations. The repair cap formed by annexins A1, A2, and A6 increased with increasing Ca2+ concentrations, while mutations in Ca2+-coordinating residues interfered with normal annexin repair cap formation. Annexin overexpression promoted the formation of external blebs at the site of membrane injury that were released from the repair cap. Overexpression of annexin A6 resulted in the formation of larger blebs being released from the repair cap. These vesicles were enriched in Ca2+-binding marker protein GCaMP5G, and this enrichment of Ca2+ correlated with a reduction of intracellular Ca2+ fluorescence near the injury site. Annexin A6 overexpression promoted membrane repair, while mutation of residue E233, a critical Ca2+-coordinating residue in annexin A6, interfered with annexin repair complex formation and decreased repair capacity. Local and systemic administration of recombinant annexin A6 reduced muscle damage in vivo. These data identified a new role for annexins in bleb release from muscle membrane lesions during membrane repair and identify annexin A6 as a therapeutic target to protect against muscle injury.
Animals. Wildtype mice from the 129T2/SvEmsJ background were bred and housed in a specific pathogen free facility on a 12-hour light/dark cycle and fed ad libitum in accordance with the Northwestern University's Institutional Animal Care and Use Committee regulations. 129T2/SvEmsJ (129T2) mice were purchased from the Jackson Laboratory (Ben Harbor, Me.; Stock #002065). mdx/hLTBP4 mice were generated as described in (Quattrocelli M, Capote J, Ohiri J C, Warner J L, Vo A H, Earley J U, et al. Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury. PLoS Genet. 2017; 13(10):e1007070; Ceco E, Bogdanovich S, Gardner B, Miller T, DeJesus A, Earley J U, et al. Targeting latent TGFbeta release in muscular dystrophy. Science translational medicine. 2014; 6(259):259ra144). Sgcg-null mice were generated as described in (Hack A A, Cordier L, Shoturma D I, Lam M Y, Sweeney H L, and McNally E M. Muscle degeneration without mechanical injury in sarcoglycan deficiency. Proceedings of the National Academy of Sciences of the United States of America. 1999; 96(19):10723-8). Two to three-month-old male and female were used for all wildtype mouse experiments. Sgcg-null cohorts were age and sex-matched with mice between 2-5 months old.
Plasmids. Plasmids encoding annexin A1, A2 and A6 with a carboxyl-terminal turboGFP tag were obtained from Origene (Rockville, Md.). Subcloning of annexin A1, A2, and A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix (Suwanee, Ga.). Site directed mutagenesis was performed by Mutagenix on annexin A1-GFP, A2-GFP and A6-GFP to create the Ca2+-binding mutants A1-D171A-GFP, A2-D161A-GFP, A6-D149A-GFP, and A6-E233A-GFP. Constructs were sequenced to verify mutagenesis. Plasmid DNA was isolated using the Qiagen endo-free Maxi prep kit (Qiagen #12362). The Ca2+ sensor GCaMP5G was purchased from (Addgene #31788).
Sequence comparison and protein schematics Protein ribbon diagrams were generated using Swiss-PdbViewer using solved crystal structures of annexin A1 (1 MCX), annexin A2 (2HYW), and annexin A6 (1AVC) available on www.rcsb.org. Clustal Omega from the European Bioinformatics Institute (EMBL-EBI) was used to align annexin sequences from www.ncbi.nlm.nih.gov annexin A1 (NM_010730; SEQ ID NO: 37), annexin A2 (NM_007585; SEQ ID NO: 38), annexin A6 (NM_013472; SEQ ID NO: 39), and annexin A6-encoding sequencing from multiple species (Homo sapiens (AAH17046.1; SEQ ID NO: 40, macaca (AFE65315.1; SEQ ID NO: 41), canis (XP_005619331.1; SEQ ID NO: 42), rattus (NP_077070.2; SEQ ID NO: 43) and mus (NP_038500.2; SEQ ID NO: 44).
Electroporation, myofiber isolation, laser injury, cap and vesicle measurement. Flexor digitorum brevis (FDB) fibers were transfected with endo-free plasmid DNA by in vivo electroporation. Methods were described previously in (Demonbreun A R, Quattrocelli M, Barefield D Y, Allen M V, Swanson K E, and McNally E M. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016; 213(6):705-18; Demonbreun A R, and McNally E M. DNA Electroporation, Isolation and Imaging of Myofibers. Journal of visualized experiments: JoVE. 2015; 106(106):e53551; DiFranco M, Quinonez M, Capote J, and Vergara J. DNA transfection of mammalian skeletal muscles using in vivo electroporation. Journal of visualized experiments: JoVE. 2009; 32(32)). Z-stack projections were acquired from consecutive acquisitions after the final time-lapse frame, approximately 4 minutes post damage, with a 0.125 μM step size between slices. Z-stack renderings were constructed in FIJI. Measurement of the cap area and feret diameter were conducted from a single slice near the middle of the z-stack using FIJI imaging tools. Fibers expressing similar levels of tagged or GCaMP5G protein were compared. GCaMP5G Ca2+ fluorescence was measured from the acquired timelapse images, using a standard rectangular ROI, placed inside the myofiber below the site of damage using FIJI. Fluorescence was expressed as F/F0. External vesicle number and GCaMP5G area were measured from endpoint z-stacks and max projection images using FIJI. Vesicles were considered external if they were found outside the sarcolemma assessed in brightfield and fluorescent channels. All measurements were acquired from myofibers isolated from at least n≥3 mice, n≥3 myofibers per mouse.
For recombinant myofibers studies, myofibers were isolated from mdx/hLTBP4 mice as described above. Myofibers were incubated in Ringer's media with or without 25 μg/ml recombinant annexin A6 (5186-A6-050, R&D systems). FM 4-64 (2.5 μm) was added to the myofibers just prior to imaging. Images were acquired and quantitated as described above. FM 4-64 area was measured using FIJI at imaging endpoint from a single slice near the middle of the z-stack. Z-stack step size (0.125 μm) was acquired from cap end to end.
Myofiber quality control was based on a number of characteristics including using adherent myofibers with intact sarcomere structure detected through brightfield imaging. Myofibers appeared devoid of tears or ruptures induced during the isolation protocol. The region of the myofiber selected for damage was linear and not located on a nucleus or neuromuscular junction. Additionally, fluorescence intensity within both the red and green channels suggested similar expression levels prior to damage.
Multiphoton laser injury and imaging. Fibers were subjected to laser-induced damage at room temperature using the Nikon A1R-MP multiphoton microscope. Imaging was performed using a 25×1.1 NA objective directed by the NIS-Elements AR imaging software. Green fluorescence protein (GFP) and FM 4-64 were excited using a 920 nm wavelength laser and emission wavelengths of 575 nm and 629 nm were collected respectively. To induce laser damage on isolated myofibers, a diffraction limited spot (diameter approximately 410 nm) was created on the lateral membrane of the myofiber using a 920 nm wavelength laser at 10-15% laser power for 1 s. Time lapse images were collected as follows: one image was collected prior to damage, one image upon damage, then every 8 seconds for 80 seconds (10 images) followed by every 30 seconds for 5 minutes (10 images). At the end of the time lapsed image series, z-stack images were collected at 250 nm intervals through the damaged site on the myofiber directed by the NIS-Elements AR imaging software. The multiphoton was used to acquire data presented in
For recombinant protein calcium studies, myofibers were isolated from wildtype mice as described above. Myofibers were incubated in 20 μg/ml recombinant annexin A6 (5186-A6-050, R&D systems) in 1 mM Ca2+ Ringers or 0 mM Ca2++EGTA. FM 1-43 (2.5 μm) was added to the myofibers just prior to imaging. Images were acquired and quantitated as described above. FM 1-43 fluorescence over time was measured using FIJI and plotted overtime as F/F0.
Cardiotoxin Injury and analysis. Tibialis anterior muscle of wildtype mice were injected with 25 μg/ml recombinant annexin A6 (5186-A6-050, R&D systems) or Ringers in sedated mice (3% isoflurane, 0.8 l/min O2). For systemic administration, wildtype mice were injected with 1 mg/kg recombinant annexin A6 (5186-A6-050, R&D systems) or PBS diluted in EBD (5 μl/g body weight) into the retro-orbital cavity of sedated mice (3% isoflurane, 0.8 l/min O2). Additionally, mice were injected with Evans' blue dye at 5 μl/g body weight (E-2129; Sigma-Aldrich, St. Louis, Mo.) dissolved in phosphate-buffered saline at 10 mg/mL cardiotoxin injury was performed injecting 20 μl of a 10 μM cardiotoxin solution in PBS in tibialis anterior or gastroc/soleus muscles in sedated animals (3% isoflurane, 0.8 l/min O2) 2 hours post pretreatment. Cardiotoxin was released down the midline of the muscle to induce a homogenous area of injury at the center of the muscle. 3 hours post cardiotoxin injection muscle was harvested.
Evans blue dye uptake. Sections (10 μm thick) from the center of frozen-embedded muscles were collected on the cryostat (chamber, −20° C.; sample, −15° C.; catalog number CM1950; Leica, Wetzlar, Germany). Tissue sections were fixed with methanol for 2 minutes, rinsed and mounted with vectashield with DAPI (H-1200, Vector Laboratories). Imaging was performed using a Zeiss Axio Observer A1 microscope (Zeiss, Oberkochen, Germany), using a 10× objective. ZEN software (Zeiss, Jena, Germany) was used for acquiring images. Fluorescence quantitation, surface plots, and muscle area were performed using FIJI (NIH). For whole tissue dye quantification, whole tissue was dissected, finely minced, weighed, and incubated at 55° C. in 1 mL of formamide for 2 hours. Spectrophotometric absorbance was measured at 620 nm.
Serum collection and CK analysis. Mice were sedated (3% isoflurane, 0.8 l/min O2) and blood was collected by means of retro-orbital puncture with heparinized capillary tubes (20-362-566; Fisher Scientific, Waltham, Mass.) into Microtainer™ Gold Top Serum Separator (365967 Becton Dickinson, Franklin Lakes, N.J.) and centrifuged at 8,000×g for 10 minutes. The plasma fractions were frozen and stored at −80° C. Serum creatine kinase (CK) was analyzed in duplicate for each mouse using the EnzyChrom Creatine Kinase Assay (ECPK-100; BioAssay Systems, Hayward, Calif.) following manufacturer's instructions as described (Demonbreun A R, Allen M V, Warner J L, Barefield D Y, Krishnan S, Swanson K E, et al. Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol. 2016; 186(6):1610-22). Results were acquired with the Synergy HTX multi-mode plate reader (BioTek®, Winooski, Vt.).
Short-term chronic dosing regimen. Sgcg-null mice were sedated (3% isoflurane, 0.8 l/min O2) and blood collected as described above. While sedated, mice were injected with 1 mg/kg recombinant annexin A6 (5186-A6-050, R&D systems) or PBS into the right retro-orbital cavity once every 3 days for a total of 5 injections and then blood drawn on day 14 post initial injection. An additional cohort of Sgcg-null mice had blood drawn as described above immediately prior to the first protein injection. Then, 1 mg/kg recombinant annexin A6 or annexin A2 (both produced by Northwestern's Protein Production Core) was injected into the right retro-orbital cavity of sedated mice, once every 3 days for a total of 5 injections and then blood drawn on day 14 post initial injection.
Exercise Injury. Sgcg-null mice were sedated (3% isoflurane, 0.8 l/min O2) and pre-exercise blood was collected by means of left retro-orbital puncture as described above. While sedated, mice were then injected 1 mg/kg recombinant annexin A6 (5186-A6-050, R&D systems) or PBS+1 mg/kg BSA into the right retro-orbital cavity at 9 am and 5 μm for 5 consecutive injection over 48 hours. Two hours post the 5th RO-injection, mice were subjected to 60 mins of treadmill running at 10 m/min at a 15° decline. Thirty minutes post exercise, blood was collected from the left retro-orbital cavity. Serum creatine kinase (CK) was analyzed as described above. Injections, exercise, and blood draws were performed blinded to treatment group.
Protein production. Recombinant mouse annexin A6 and mouse annexin A2 were produced and purified by the Northwestern's recombinant protein production core. Briefly, mouse annexin A6 (MG222645, Origene, Rockville, Md.) and annexin A2 (MG205064, Origene, Rockville, Md.) were subcloned into the pCMV6-AC-HIS backbone (PS100002, Origene, Rockville, Md.). Plasmid was transfected and expressed with ExpiCHO expression system (A29133, ThermoFisher Scientific, Waltham, Mass.). Carboxy-terminally tagged recombinant protein was purified with Ni-charged MagBeads (L00295; GenScript, Piscataway, N.J.) and purity evaluated through SDS-Page. Protein purity was additionally validated through immunoblot using anti-HIS (MAB050; R&D Systems, Minneapolis, Minn.) and anti-annexin A6 (ab31026, Abcam) or anti-annexin A2 (ab154113, Abcam) antibodies. Protein was diluted in phosphate buffered saline and stored at −80° C.
Calcium kinetics. FDB muscle was electroporated and isolated as described above. Myofibers were damaged in Ringers solution with Ca2+ concentrations of 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.175 mM, 0.1 mM, 0.050 mM, and 0 mM. EDTA was added as a Ca2+ chelating agent in only in the 0 mM Ca2+ Ringers. Myofibers were isolated directly into 2 mM, 1 mM and 0.5 mM Ringers for those experiments respectively. For experiments using less than 0.5 mM Ca2+ myofibers were isolated in 0.5 mM Ca2+ Ringers and then diluted with 0 mM EDTA-free Ca2+ Ringers. For 0 mM experiments, myofibers were isolated in 0.5 mM Ca2+ Ringers and then replaced with 0 mM Ca2++EDTA Ringers just prior to imaging. Co-electroporation of wildtype annexin+wildtype annexin constructs was performed in one mouse foot, while the contralateral foot was co-electroporated with wildtype annexin+mutant annexin. All measurements were acquired from myofibers isolated from at least n≥2 mice, n≥3 myofibers per mouse at each Ca2+ concentration. Area-Ca2+ curves were fitted with a Hill Curve at Ca2+ concentrations ranging from 0-2 mM. Kinetic parameters were calculated using Prism Graphpad.
Calcium and pH indicator dye measurements. Wildtype FDBs were isolated and plated in Ringers on Matek glass-bottom dishes as described above. Twenty minutes prior to imaging, myofibers were loaded with Fluo-4 AM at 37° C. (F10489, ThermoFisher Scientific, Waltham, Mass.) or pHrodo AM (P35373, ThermoFisher Scientific, Waltham, Mass.) as described in the instruction manual. Fibers were rinsed once with Ringers then subsequently damaged and imaged on the Nikon A1R GaSP, as described above. Fluorescence intensity was measured using FIJI (NIH). phRodo change in fluorescence intensity was calculated as F/F0. Data was acquired from n=3 mice per experiment from multiple myofibers per mouse. Additionally, wildtype myofibers were incubated in 0 mM Ca2+ for 1 hour, preloaded with Fluo-4 AM for 20 minutes prior to imaging, rinsed and then damaged on the MP+ in the presence of 0 mM external Ca2+ with or without EGTA in the external Ringers Solution.
Cardiotoxin injection and histology. Wildtype mice were sedated (3% isoflurane, 0.8 l/min O2) and then injected 1 mg/kg recombinant annexin A6 (5186-A6-050, R&D systems) or PBS+1 mg/kg BSA into the right retro-orbital cavity and allowed to recover. Two hours post injection mice were sedated for a second time. While sedated the tibialis anterior muscles were injected along the midline with 10 μm cardiotoxin in 20 ul of PBS as described above. Seven days post injection, the muscle was isolated and frozen. Muscle sections were acquired every 100 μm from muscle tendon into the mid belly, fixed and stained with hematoxylin and eosin. Imaging was performed using a Zeiss Axio Observer A1 microscope (Zeiss, Oberkochen, Germany), using a 10× objective. ZEN software (Zeiss, Jena, Germany) was used for acquiring tiled images. Percent injury area was calculated as the average injured area (containing internal myonuclei) divided by total muscle area of 3 sections per muscle. One muscle from each group was excluded due to technical error in tissue processing. Damage area was measured using FIJI (NIH).
Single cell Ca2+ and shortening measurements. Isolated FDB fibers were plated on laminin coated glass-bottomed 35 mm dishes for one hour and then cultured overnight in DMEM with 10% FBS and 1% penicillin/streptomycin at 37° C. in a 10% CO2 incubator. One hour prior to data acquisition, the medium was removed and cells were incubated in Tyrode buffer (119 mM NaCl, 5 mM KCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2) with 10 μM Indo-1 AM (TefLabs) for 1 hour at 37° C. in a 10% CO2 incubator. Dishes were then filled with Tyrode buffer, mounted on a custom stage and platinum pacing electrodes were inserted into the dish. Stimulation was elicited using a 701C high-powered stimulator controlled by the 950A software (Aurora Scientific). Stimulation was performed at 40 and 80 Hz, 5 ms pulse width, 100 ms duration. Ratiometric Ca2+ signals were collected with two photomultiplier tubes and a FluoroDaq controller. Video sarcomere length was recorded with a high-speed camera and fast Fourier transform using the Aurora Scientific 900B-VSL system (Aurora, Ontario). Ten transients were collected over 20 seconds and averaged together per cell per frequency.
Statistical analysis. Statistical analyses were performed with Prism (Graphpad, La Jolla, Calif.). Comparisons relied on ANOVA ((1way ANOVA for 1 variable, 2way ANOVA for two variables (typically area and Ca2+ concentration)). Otherwise, unpaired two-tailed t-tests were performed. P-values of less than or equal to 0.05 were considered significant. Error bars represent +/−standard error of the mean (SEM).
Study approval. The study was conducted with the approval of Northwestern University's Institutional Animal Care and Use Committee (Chicago, Ill.).
Ca2+ localizes to the repair cap upon membrane damage. Activation of muscle membrane repair requires the presence of external Ca2+ (Bansal D, Miyake K, Vogel S S, Groh S, Chen C C, Williamson R, et al. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature. 2003; 423(6936):168-72). Annexin proteins have previously been shown to aggregate into repair caps at the site of injury bordered by annexin-free zone within the cytoplasm under the repair cap (Demonbreun A R, Quattrocelli M, Barefield D Y, Allen M V, Swanson K E, and McNally E M. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016; 213(6):705-18; Demonbreun A R, Allen M V, Warner J L, Barefield D Y, Krishnan S, Swanson K E, et al. Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol. 2016; 186(6):1610-22; Swaggart K A, Demonbreun A R, Vo A H, Swanson K E, Kim E Y, Fahrenbach J P, et al. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair. Proceedings of the National Academy of Sciences of the United States of America. 2014; 111(16):6004-9; Quattrocelli M, Barefield D Y, Warner J L, Vo A H, Hadhazy M, Earley J U, et al. Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy. J Clin Invest. 2017; 127(6):2418-32). To visualize Ca2+ dynamics at the site of injury in real-time, an in vivo fluorescent Ca2+ indicator protein, GCaMP5G, was utilized. GCaMP5G is a fusion protein composed of green fluorescent protein (GFP), the calcium-binding protein calmodulin, and the calmodulin M13 binding peptide. GCaMP5G has minimal fluorescence when not bound to Ca2+, and Ca2+ binding results in a conformational change within the protein increasing the fluorescence intensity of GFP (Akerboom J, Chen T W, Wardill T J, Tian L, Marvin J S, Mutlu S, et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci. 2012; 32(40):13819-40). Wildtype flexor digitorum brevis (FDB) muscle was electroporated with the GCaMP5G plasmid and then injured the plasma membrane using laser ablation (Demonbreun A R, Quattrocelli M, Barefield D Y, Allen M V, Swanson K E, and McNally E M. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016; 213(6):705-18; Demonbreun A R, and McNally E M. DNA Electroporation, Isolation and Imaging of Myofibers. Journal of visualized experiments: JoVE. 2015; 106(106):e53551). Within two seconds of membrane injury (arrow), GCaMP5G fluorescence accumulated in the cytoplasm at the site of injury. GCaMP5G fluorescence intensity progressively increased through 260 seconds of imaging (
To ensure these results were not a reflection of protein aggregation of the GCaMP5G sensor, wildtype myofibers were injured in the presence of Fluo-4 AM. Fluo-4 AM is a non-protein, Ca2+ indicator dye that increases fluorescence intensity upon binding Ca2+ and is routinely used to measure Ca2+ dynamics. Similar to GCaMP5G fluorescence, Fluo-4 AM fluorescence intensity increased at the site of laser-induced membrane injury 2 seconds post damage (arrow) and continued to increase intensity through the 260 seconds of imaging (
Annexin repair caps exhibit differential Ca2+ sensitivity during repair cap recruitment. Annexin proteins are Ca2+-dependent phospholipid and actin binding proteins that contain four annexin repeat domains or eight in the case of annexin A6 (
Annexin overexpression promotes bleb formation at the site of membrane injury. Membrane repair studies in Lytechinus pictus and Xenopus oocytes have observed membranous structures emerging and erupting from the site of membrane repair (Davenport N R, Sonnemann K J, Eliceiri K W, and Bement W M. Membrane dynamics during cellular wound repair. Mol Biol Cell. 2016; 27(14):2272-85; Bi G Q, Alderton J M, and Steinhardt R A. Calcium-regulated exocytosis is required for cell membrane resealing. The Journal of cell biology. 1995; 131(6 Pt 2):1747-58). Additionally, in artificial membrane preparations, the addition of recombinant annexins induced membrane folding or blebbing in a Ca2+-dependent manner at sites of membrane imperfection (Boye T L, Jeppesen J C, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018; 8(1):10309; Boye T L, Maeda K, Pezeshkian W, Sonder S L, Haeger S C, Gerke V, et al. Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair. Nat Commun. 2017; 8(1):1623). It was investigated whether similar findings could be observed at the site of muscle membrane injury in live skeletal myofibers. GCaMP5G was expressed alone or in combination with annexin A1, A2 or A6 in skeletal myofibers. It was found that overexpression of annexins promoted the formation of extracellular blebs emanating from annexin repair caps at the membrane lesion (
Decreased intracellular Ca2+ fluorescence at the site of injury with annexin overexpression. Time lapse imaging of the Ca2+ indicator GCaMP5G after laser injury suggested intracellular Ca2+ was decreasing concomitant with extracellular bleb formation suggesting that these blebs serve to reduce intracellular Ca2+ accumulation through excretion. The annexin-induced reduction in intracellular Ca2+ fluorescence could be seen for all three annexins A1, A2 and A6, but was most evident for annexin A2 and A6 (
Overexpression of Ca2+-binding proteins like annexins may have unexpected effects on intracellular Ca2+ signaling and cellular function. Therefore, the Ca2+ handling and contractile properties of isolated myofibers overexpressing annexin A6 was evaluated compared to controls. Isolated myofibers expressing annexin A6 were loaded with the ratiometric Ca2+ indicator dye Indo-1, and we observed no differences in Ca2+ cycling at 40 or 80 Hz stimulation frequencies between annexin A6 or control fibers (
Annexin A6 Ca2+ binding is required for repair cap formation and myofiber repair. Mutation of annexin A1 residue D171 and annexin A2 residue D161 were previously shown to inhibit annexin membrane translocation in HEK cells (McNeil A K, Rescher U, Gerke V, and McNeil P L. Requirement for annexin A1 in plasma membrane repair. The Journal of biological chemistry. 2006; 281(46):35202-7; Jost M, Thiel C, Weber K, and Gerke V. Mapping of three unique Ca(2+)-binding sites in human annexin II. Eur J Biochem. 1992; 207(3):923-30). It was queried whether these mutations would inhibit translocation and formation of the macromolecular annexin repair cap formed after muscle membrane injury in live myofibers. Alignment of annexins A1, A2 and A6 protein sequences was used to identify the conserved residues within the consensus sequence of type II Ca2+ binding sites across all three annexin proteins (
Ca2+-binding of both annexin A1 and A2 was also required for repair cap formation. A1 D171A and A2D161A mutant cap size was reduced compared to wildtype annexin A1 and A2 controls, respectively. Expression of mutant annexin A1 D171A and A2D161A significantly reduced the repair cap diameter (DMAX) of the respective co-expressed wildtype annexin protein (
To determine the effect of dominant negative annexin A6 on the assembly of annexins A1, A2, and A6 at the repair cap and membrane repair capacity, laser injury was similarly performed on isolated myofibers in the presence of FM 4-64. Myofibers expressing annexin A6E233A-GFP had increased FM 4-64 fluorescence area after laser injury compared to control myofibers expressing wildtype annexin A6-GFP (
Annexin A6 protected against laser-induced myofiber injury in vitro in a Ca2-dependent manner. Since annexin A6 facilitates the formation of the macromolecular repair cap complex and was the most efficient at forming large, Ca2+-filled blebs at the site of membrane injury, it was assessed whether expression of annexin A6 would reduce membrane injury in wildtype myofibers. Wildtype myofibers were electroporated with annexin A6-GFP or mock electroporated and then laser damaged in the presence of FM 4-64 to mark the injury area. Wildtype myofibers overexpressing annexin A6 had decreased FM 4-64 dye uptake after laser-induced membrane injury compared to control myofibers (
Since intracellular annexin A6 targets phospholipids exposed at the site of membrane injury and enhances membrane repair capacity, we hypothesized that extracellular recombinant annexin A6 (rANXA6) would also localize to the site of injury and protect against membrane injury. Muscular dystrophy is a progressive muscle wasting disease, arising from loss-of-function mutations in critical cytoskeletal or membrane-associated proteins, often resulting in fragile plasma membranes. To determine if recombinant annexin A6 could protect against membrane insult in both healthy and dystrophic muscle, wildtype and dystrophic myofibers from a model representing Duchenne Muscular Dystrophy were isolated and incubated with recombinant annexin A6 or vehicle control. Laser injury was conducted in the presence of FM 4-64 to visualize the extent of injury. Treatment with extracellular recombinant annexin A6 reduced FM 4-64 fluorescence area compared to vehicle control-treated myofibers, indicating enhanced repair in both healthy and dystrophic myofibers (
To assess whether recombinant annexin A6's protective effects required external Ca2+, wildtype myofibers were pretreated with recombinant annexin A6, loaded with FM 1-43, a fluorescence marker of membrane damage similar to FM4-64. and subsequently damaged in solution containing 1 mM Ca2+ or 0 mM Ca2++EGTA, a Ca2+ chelator. FM 1-43 fluorescence accumulation at the lesion over time (F/F0) was significantly increased in the absence of Ca2+ compared to in the presence of 1 mM Ca2+ (
Recombinant annexin A6 protected against acute muscle injury in vivo. To determine the therapeutic potential of recombinant annexin A6 to protect against muscle injury in vivo, recombinant annexin A6 was utilized as a tool compound. Recombinant annexin A6 or vehicle control was injected intramuscularly into the tibialis anterior (TA) muscles of wildtype mice two hours prior to toxin-induced injury. Mice were injected intraperitoneally with Evan's blue dye, a vital tracer that is excluded by intact healthy myofibers but is readily taken up in injured permeable myofibers (Jennische E, and Hansson H A. Postischemic skeletal muscle injury: patterns of injury in relation to adequacy of reperfusion. Exp Mol Pathol. 1986; 44(3):272-80). Three hours post-cardiotoxin injury muscle was evaluated for Evan's blue dye uptake (
Although intramuscular injection of annexin A6 was effective at reducing acute injury, this route of application is not optimal for large muscle groups, internal tissues, or treatment of chronic diseases. Therefore, the efficacy of recombinant annexin A6 administered systemically via retro-orbital (RO) injection in the protection against acute muscle injury was examined. Recombinant annexin A6 or vehicle was injected into the retro-orbital cavity two hours prior to toxin-induced injury. Mice were simultaneously injected with Evan's blue dye. Three hours post-cardiotoxin injury muscle was evaluated for Evan's blue dye uptake (
To determine if recombinant annexin A6 administration enhanced myocyte survival and/or recovery from injury, mice were administered recombinant annexin A6 or BSA control, at 1 mg/kg, through retro-orbital systemic injection. Two hours post protein administration, cardiotoxin was injected into the tibialis anterior muscles to induce focal muscle injury. Muscle was harvested 7 days post injury and histology evaluated for injury area (
Recombinant annexin A6 protected against chronic muscle injury in vivo. The ability of recombinant annexin A6, administered systemically, to protect against muscle damage in the Sgcg-null mouse model of Limb Girdle Muscular Dystrophy type 2C (LGMD2C) (Hack A A, Cordier L, Shoturma D I, Lam M Y, Sweeney H L, and McNally E M. Muscle degeneration without mechanical injury in sarcoglycan deficiency. Proceedings of the National Academy of Sciences of the United States of America. 1999; 96(19):10723-8) was assessed next. Sgcg-null mice lack γ-sarcoglycan, an integral membrane component of the dystrophin glycoprotein complex required for membrane stability and function. Humans and mice lacking γ-sarcoglycan develop progressive muscle disease, reduced muscle function and elevated serum creatine kinase (CK), a serum biomarker of muscle injury and membrane leak. To determine if recombinant annexin A6 protected in dystrophic muscle, Sgcg-null mice were treated systemically with recombinant annexin A6 or BSA control over 48 hours (
Annexins promote calcium-filled bleb formation at the site of membrane injury. Plasma membrane instability is inherent to many forms of muscular dystrophy and thought to contribute to dysregulated Ca2+ homoeostasis and disease pathogenesis. Molkentin and colleagues showed that transgenic overexpression of TRPC3 was sufficient to increase myofiber Ca2+ influx and result in a dystrophy-like phenotype (Millay D P, Goonasekera S A, Sargent M A, Maillet M, Aronow B J, and Molkentin J D. Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism. Proceedings of the National Academy of Sciences of the United States of America. 2009; 106(45):19023-8). Correspondingly, transgenic overexpression of SERCA1 reduced cytosolic Ca2+ levels and mitigated dystrophic muscle pathology implicating Ca2+ in disease progression (Goonasekera S A, Lam C K, Millay D P, Sargent M A, Hajjar R J, Kranias E G, et al. Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle. J Clin Invest. 2011; 121(3):1044-52). As shown and described herein, increased expression of annexins in muscle fibers decreased injury-associated Ca2+ fluorescence within myofibers. This reduction of Ca2+-associated fluorescence was at the injury site and correlated with extracellular bleb formation emanating from annexin repair caps. Both annexin A2 or A6 could induce the formation of membranous blebs containing the Ca2+-binding protein GCaMP5G. Furthermore, overexpression of annexins A1, A2, and A6 each reduced endpoint Ca2+ fluorescence accumulation within the myofiber after injury. Of the three annexins tested, annexin A6 overexpression resulted in the most sustained effect on reducing injury-associated Ca2+ accumulation inducing the formation of large GCaMP5G-containing blebs. In HEK293 cells damaged with streptolysin O (SLO), the presence of extracellular membranous blebs correlated with increased cell survival and reduction in cytoplasmic Ca2+ levels, a process facilitated by annexin A1 (Babiychuk E B, Monastyrskaya K, Potez S, and Draeger A. Blebbing confers resistance against cell lysis. Cell Death Differ. 2011; 18(1):80-9). Davenport and colleagues showed overexpression of annexin A1-GFP in injured Xenopus oocytes resulted in annexin A1 positive blebs originating from the site of damage (Davenport N R, Sonnemann K J, Eliceiri K W, and Bement W M. Membrane dynamics during cellular wound repair. Mol Biol Cell. 2016; 27(14):2272-85). However, the effects of annexins A2 or A6 overexpression were not assessed in either study. These data combined suggest bleb formation as a mechanism of membrane repair is conserved across species and tissue types and is facilitated by the presence of annexin proteins.
Without being bound by theory, it is contemplated that annexin A6 facilitates cytoplasmic Ca2+ and protein excretion into extracellular blebs whose formation is further induced by annexin A1 and annexin A2. In artificial membrane patches, the presence of annexin A1 or annexin A2 induced bleb formation at sites of membrane imperfection (Boye T L, Jeppesen J C, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018; 8(1):10309). In contrast, the presence of annexin A6 induced Ca2+-dependent contraction of artificial membrane into large folds (Boye T L, Jeppesen J C, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018; 8(1):10309). The difference between annexin A6 inducing blebs in live myofibers or folds in artificial membrane may reflect the presence of endogenously expressed annexin A1 and A2 in isolated myofibers compared with exposure to single recombinant annexin protein in the artificial membrane studies. Without being bound by theory, it is contemplated that within the macromolecular repair complex, multiple annexins actively participate in bleb formation, which acts to remove large membrane lesions facilitating wound closure, excision of damaged membrane, and reduction of Ca2+ at the injury site.
Annexin A6 protects against muscle membrane injury and enhances membrane repair. We showed that annexin proteins, including annexin A1, A2, and A6, localize to the site of membrane injury facilitating membrane repair cap and bleb formation. Mutation of annexin A6 abrogated repair cap formation, decreasing repair capacity, resulting in increased dye uptake. On the other hand, pretreatment with recombinant annexin A6 reduced dye uptake after laser-induced muscle injury and after toxin-induced muscle injury in vivo. As a therapeutic tool, enhancing the cells' ability to repair and/or reduce injury through stabilizing the cell membrane are both beneficial avenues that can lead to improved cell survival. Previous studies have shown that annexin A6 is upregulated in muscle from models of chronic muscular dystrophy (Demonbreun A R, Allen M V, Warner J L, Barefield D Y, Krishnan S, Swanson K E, et al. Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption. Am J Pathol. 2016; 186(6):1610-22; Swaggart K A, Demonbreun A R, Vo A H, Swanson K E, Kim E Y, Fahrenbach J P, et al. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair. Proceedings of the National Academy of Sciences of the United States of America. 2014; 111(16):6004-9; Demonbreun A R, Rossi A E, Alvarez M G, Swanson K E, Deveaux H K, Earley J U, et al. Dysferlin and myoferlin regulate transverse tubule formation and glycerol sensitivity. Am J Pathol. 2014; 184(1):248-59). Proteomic profiling of mdx mouse muscle showed that annexins A1 and A2 are enriched in mdx muscle membrane, consistent with a role for annexins at the membrane of injured muscle cells (Murphy S, Zweyer M, Henry M, Meleady P, Mundegar R R, Swandulla D, et al. Proteomic analysis of the sarcolemma-enriched fraction from dystrophic mdx-4cv skeletal muscle. J Proteomics. 2018). Annexins bind membrane phospholipids, including phosphatidylserine, which is exposed during membrane disruption. Phosphatidylserine rearrangement after injury provides an optimal binding target for extracellular annexins to facilitate membrane folding, blebbing, and rolling at sites of membrane damage and imperfection (Boye T L, Jeppesen J C, Maeda K, Pezeshkian W, Solovyeva V, Nylandsted J, et al. Annexins induce curvature on free-edge membranes displaying distinct morphologies. Sci Rep. 2018; 8(1):10309). Upregulation of annexins is contemplated to be a compensatory mechanism to facilitate excision of defective membrane in fibers undergoing chronic damage.
Additional studies of cardiac muscle injury further suggest a role for annexin proteins in modulating the repair response. Administration of recombinant annexin A1 or the N-terminal annexin A1 peptide (AC2-26) elicited a cardioprotective response in a rat model of myocardial ischemia-reperfusion-induced injury (La M, D'Amico M, Bandiera S, Di Filippo C, Oliani S M, Gavins F N, et al. Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action. FASEB J. 2001; 15(12):2247-56). Meng et al demonstrated that downregulation of annexin A3 resulted in cardioprotection, decreasing rat myocardial infarct size through activation of AKT signaling (Meng H, Zhang Y, An ST, and Chen Y. Annexin A3 gene silencing promotes myocardial cell repair through activation of the PI3K/Akt signaling pathway in rats with acute myocardial infarction. J Cell Physiol. 2019; 234(7):10535-46). In addition to membrane reorganization, annexins act as scaffolds regulating multiple downstream intracellular signaling cascades important for orchestrating repair from injury. Both intra- and extra-cellular functions of annexin proteins should be considered when evaluating the therapeutic potential of annexin proteins.
The studies presented herein indicate that human recombinant annexin A6 protein is a suitable biologic to protect against acute muscle injury. It is shown herein that human recombinant annexin A6 was capable of resealing injured membrane in mouse models, confirming functional activity of the human recombinant protein in a mouse preclinical model. Human and mouse annexin A6 proteins are 94.35% identical at the amino acid level with increasing percent amino acid conservation between humans and rat (94.65%), dog (95.54%), and monkey (98.96%), and this high degree of similarly is consistent with human recombinant protein having efficacy in a mouse model (
Combinatorial approaches to improve membrane repair. Recombinant annexin A6 and its ability to protect normal and dystrophic muscle from laser-induced membrane injury is described and shown herein. In addition, both intramuscular and systemic administration of recombinant annexin A6 protected against toxin-induced muscle membrane injury in vivo. It was previously found that glucocorticoid administration increased annexin expression in muscle, and this correlated with enhanced muscle repair in multiple mouse models of muscular dystrophy including mdx (DMD), dysferlin-null (Limb Girdle Muscular Dystrophy 2B), and γ-sarcoglycan-null (Limb Girdle Muscular Dystrophy 2C) mice (Quattrocelli M, Barefield D Y, Warner J L, Vo A H, Hadhazy M, Earley J U, et al. Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy. J Clin Invest. 2017; 127(6):2418-32; Quattrocelli M, Salamone I M, Page P G, Warner J L, Demonbreun A R, and McNally E M. Intermittent Glucocorticoid Dosing Improves Muscle Repair and Function in Mice with Limb-Girdle Muscular Dystrophy. Am J Pathol. 2017; 187(11):2520-35). Glucocorticoid treatment also increased the expression of the Trim72 gene that encodes mitsugumin 53 (known as MG53), a repair protein that localizes to the site of membrane injury and considered a “molecular band-aid” for improving cellular wound healing. Similar to the annexins, MG53 is upregulated in chronic muscle injury and enhances repair in dystrophic muscles, as well as other tissues like heart, lung, kidney (Waddell L B, Lemckert F A, Zheng X F, Tran J, Evesson F J, Hawkes J M, et al. Dysferlin, annexin A1, and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch. J Neuropathol Exp Neurol. 2011; 70(4):302-13; Duann P, Li H, Lin P, Tan T, Wang Z, Chen K, et al. MG53-mediated cell membrane repair protects against acute kidney injury. Science translational medicine. 2015; 7(279):279ra36; He B, Tang R H, Weisleder N, Xiao B, Yuan Z, Cai C, et al. Enhancing muscle membrane repair by gene delivery of MG53 ameliorates muscular dystrophy and heart failure in delta-Sarcoglycan-deficient hamsters. Molecular therapy: the journal of the American Society of Gene Therapy. 2012; 20(4):727-35; Jia Y, Chen K, Lin P, Lieber G, Nishi M, Yan R, et al. Treatment of acute lung injury by targeting MG53-mediated cell membrane repair. Nat Commun. 2014; 5:4387; Liu J, Zhu H, Zheng Y, Xu Z, Li L, Tan T, et al. Cardioprotection of recombinant human MG53 protein in a porcine model of ischemia and reperfusion injury. Journal of molecular and cellular cardiology. 2015; 80:10-9; Weisleder N, Takizawa N, Lin P, Wang X, Cao C, Zhang Y, et al. Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy. Science translational medicine. 2012; 4(139):139ra85). MG53 is a component of the annexin-mediated repair complex, localizing adjacent to the annexin repair cap (Demonbreun A R, Quattrocelli M, Barefield D Y, Allen M V, Swanson K E, and McNally E M. An actin-dependent annexin complex mediates plasma membrane repair in muscle. The Journal of cell biology. 2016; 213(6):705-18).
Additional experiments were designed and conducted to evaluate the role of endogenous annexin A6, annexin A6 in heart, and annexin A6 in additional models of muscular dystrophy.
Animals. Genetically-encoded annexin A6GFP mice were generated and backcrossed onto the 129T2/SvEmsJ background. Dysferlin-null mice on the 129T2/SvEmsJ background were previously generated (Demonbreun et al. HMG 2011). Sgcg-null mice were generated as described in (Hack et al PNAS 1999). Mice were bred and housed in a specific pathogen free facility on a 12-hour light/dark cycle and fed ad libitum in accordance with the Northwestern University's Institutional Animal Care and Use Committee regulations. 129T2/SvEmsJ (129T2) mice were originally purchased from the Jackson Laboratory (Ben Harbor, Me.; Stock #002065). Two to three-month-old male and female were used for all experiments. All animal experiments were approved in accordance with the Northwestern University's Institutional Animal Care and Use Committee regulations.
Genomic structure schematic. Annexin A6 genomic structure was visualized using UCSC genome browser.
Myofiber isolation. Flexor digitorum brevis (FDB) fibers were isolated with methods that were described previously in (Demonbreun and McNally, 2015; Demonbreun et al., 2016b; DiFranco et al., 2009). Briefly, fibers were dissociated in 0.2% BSA plus collagenase type II (Cat #17101, Thermo Fisher Scientific, Waltham, Mass.) for 60 minutes at 37 degrees in 10% CO2. Fibers were then moved to Ringers solution and placed on MatTek confocal microscopy dishes (Cat #P35G-1.5-14-C, MatTek, Ashland Mass.).
Immunofluorescence microscopy. Mice were systemically injected with recombinant annexin A6 with a C-terminal HIS tag (R&D). Muscle was harvested and flash frozen. Muscle sections (10 μm thick) from the center of frozen-embedded muscles were collected on the cryostat (chamber, −20° C.; sample, −15° C.; catalog number CM1950; Leica, Wetzlar, Germany) for immunostaining. Tissues were fixed with 4% paraformaldehyde for 10 minutes on ice. Block and permeabilization were with 0.1% Triton (catalog number X-100; Sigma-Aldrich), 10% fetal bovine serum, and PBS for 60 minutes. For laminin detection, anti-laminin antibody was used at a dilution of 1:100 and for HIS detection, anti-HIS antibody was used at 1:100 overnight at 4° C. Hoechst labelled nuclei. Sections were PBS rinsed, incubated with secondary antibody for 1 hour, PBS rinsed, and mounted. Imaging was performed using a Zeiss Axio Observer A1 microscope (Zeiss, Oberkochen, Germany).
Cardiomyocyte isolation. Mice were treated with 50 U heparin intraperitoneally 20 minutes before sacrifice. Mice were anesthetized under 5% vaporized isoflurane mixed with 100% oxygen. A thoracotomy was performed and the heart and lungs rapidly excised and submerged into ice-cold Tyrode solution without calcium (143-mM NaCl, 2.5-mM KCl, 16-mM MgCl2, 11-mM glucose, 25-mM NaHCO3, pH adjusted to 7.4). The ascending aorta was dissected out of the surrounding tissue and cannulated with an animal feeding needle (7900, Cadence Science, Staunton, Va.) and secured with a 6-0 silk suture. The heart was initially perfused with 1 ml of ice-cold calcium-free Tyrode solution before being transferred to a Langendorff apparatus (Radnoti, Covina, Calif.). Hearts were perfused with 37° C. calcium-free Tyrode solution using a constant pressure (65-cm vertical distance between the buffer reservoir and cannula tip) for 1 to 2 minutes before perfusion for 5.5 minutes with digestion solution (0.15% collagenase type 2 [Worthington Biochemical, Lakewood, New Jersey], 0.1% 2,3-butanedione monoxime, 0.1% glucose, 100-U/ml penicillin/streptomycin, 112-mM NaCl, 4.7-mM KCl, 0.6-mM KH2PO4, 40-μM CaCl2, 0.6-mM Na2HPO4, 1.2-mM MgSO4, 30-μM phenol red, 21.4-mM NaHCO3, 10-mM HEPES, and 30-mM taurine; pH adjusted to 7.4). The heart was removed from the cannula, triturated with a transfer pipette, and filtered through a 100-μm cell strainer. Cardiomyocytes were allowed to pellet by gravity for 7 minutes, followed by aspiration of digestion media and washing with stop buffer (formulated identically to digestion solution except with no collagenase and with 1% bovine serum albumin). Cells were again allowed to gravity pellet followed by a wash in stop buffer without bovine serum albumin. Cardiomyocytes were tolerated to calcium by adding Tyrode buffer with 0.3-mM CaCl2 dropwise. Cell culture dishes were coated with 20 μg/ml laminin (23017-015; Gibco, Thermo Fisher Scientific, Waltham, Mass.) for 1 hour at room temperature. Laminin solution was aspirated followed by plating of cardiomyocytes for 1 hour to allow cell adhesion before experimentation.
Multiphoton laser injury and imaging. Isolated fibers were subjected to laser-induced damage at room temperature using the Nikon A1R-MP multiphoton microscope. Imaging was performed using a 25×1.1 NA objective directed by the NIS-Elements AR imaging software. Green fluorescence protein (GFP) and FM 4-64 were excited using a 920 nm wavelength laser and emission wavelengths of 575 nm and 629 nm were collected respectively. To induce laser damage on isolated myofibers, a diffraction limited spot (diameter approximately 410 nm) was created on the lateral membrane of the myofiber using a 920 nm wavelength laser at 10-15% laser power for 1 second. Time lapse images were collected as follows: one image was collected prior to damage, one image upon damage, then every 8 s for 80 s (10 images) followed by every 30 seconds for 5 min (10 images). At the end of the time lapsed image series, z-stack images were collected at 250 nm intervals through the damaged site on the myofiber directed by the NIS-Elements AR imaging software. Fluorescence intensity and cap area were measured using Fiji (NIH).
For recombinant protein studies, myofibers were isolated from mice as described above. Myofibers were incubated in 10-40 μg/ml recombinant annexin A6 (5186-A6-050, R&D systems) in 1 mM Ca2+ Ringers or BSA control. Cap size was assessed from acquired images in FIJI. FM 4-64 (2.5 μm) was added to the myofibers just prior to imaging. Images were acquired and quantitated as described above. FM 4-64 fluorescence at endpoint was measured using FIJI.
For prednisone studies, prednisone (catalog P6254) was resuspended in DMSO (catalog D2650, Sigma-Aldrich) at 5 mg/ml. Dosing was based on pretreatment weights (1 mg/kg body weight) in 30 μl total PBS. Mice were injected at 7 am on the day prior to imaging. On injection days, stock solutions stored at −20° C. were diluted into sterile Eppendorf tubes containing sterile PBS (catalog 14190, Life Technologies). Sterile BD Micro-Fine IV Insulin Syringes (catalog 14-829-1A, Fisher Scientific) were used to inject the I.P. cavity of non-sedated animals. 24 hours post injection myofibers were harvest and then imaged the same day.
Statistical analysis. Statistical analyses were performed with Prism (Graphpad, La Jolla, Calif.). Comparisons relied on ANOVA (1way ANOVA for 1 variable). Otherwise, unpaired two-tailed t-tests were performed. P value less than or equal to 0.05 was considered significant. Data were presented as single values were appropriate. Error bars represent +/−standard error of the mean (SEM).
Results of the experiments are shown in
Glucocorticoid steroids modify endogenous annexin A6GFP localization, protecting against insult. Gene editing was used to introduce a GFP tag at the carboxyl terminus of the Anxa6 locus in mice to create Anxa6em1(GFP) mice. These mice offer the advantage of examining genomically-encoded annexin A6 under the control of the Anxa6 promoter and regulatory sequences. Because Anxa6em1(GFP) mice express annexin A6 protein under the control of the Anxa6 gene, expression levels are at normal to lower levels than plasmid encoded annexin A6-GFP. This animal model allows for the examination of a range of annexin A6 protein expression in real time. Glucocorticoid steroids, including prednisone, have been shown to upregulate annexin A1 and A6 expression. Furthermore, prednisone administration protected against membrane injury, reducing plasmid overexpressed annexin GFP cap formation after laser injury. To determine if prednisone modulated genomically-encoded annexin A6GFP cap kinetics, Anxa6em1(GFP) mice were injected with 1 mg/kg prednisone or control DMSO into the intraperitoneal cavity. Twenty-four hours post injection, myofibers were isolated and subjected to laser injury. Annexin A6GFP cap size was reduced in myofibers treated with prednisone compared to controls (
Recombinant annexin A6 promotes skeletal muscle repair. It was shown that recombinant annexin A6 protected against laser-induced injury by reducing FM dye uptake in healthy and dystrophic myofibers. To further elucidate the mechanism by which recombinant annexin A6 confers protection, Anxa6em1(GFP) myofibers were pretreated with recombinant annexin A6 protein or control. Subsequently, myofibers were incubated in FM 4-64 dye and subjected to laser injury. Myofibers pretreated with recombinant annexin A6 had smaller annexin A6GFP repair caps and a concomitant reduction in FM dye uptake, compared to control myofibers (
Annexin A6GFP is expressed in the heart and forms repair caps in injured cardiomyocytes. Annexin A6 was identified as a modifier of membrane leak in the heart, however, methods to evaluate cardiomyocyte repair were previously lacking. It was hypothesized that if endogenous annexin A6GFP was expressed in the heart that this new Anxa6em1(GFP) mouse model could be used as a tool to evaluate endogenous cardiomyocyte membrane repair. To determine if endogenous annexin A6GFP played a role in membrane repair in tissues beyond skeletal muscle, Anxa6em1(GFP) cardiomyocytes were isolated and subjected to laser injury. Endogenous annexin A6GFP localized in a sarcomeric pattern in live, isolated cardiomyocytes, similar to skeletal myofibers (
Annexin A6 enhances repair in a model of Limb Girdle Muscular Dystrophy 2B (LGMD2B). Dysferlin is a Ca2+-binding, membrane-associated protein implicated in membrane repair. Loss-of-function mutations is dysferlin reduce membrane repair capacity resulting in Limb Girdle Muscular Dystrophy 2B (LGMD2B). It was hypothesized that agents that enhance repair or reduce injury susceptibility could provide therapeutic benefit for treating LGMD2B. To test this hypothesis, mice lacking dysferlin on the 129 background (Dysf129) were electroporated with annexin A6 plasmid and then were subjected to laser injury in the presence of FM 4-64. Myofibers overexpressing annexin A6 had a significant reduction in FM 4-64 dye uptake after injury (
Recombinant annexin A6 localizes to the sarcolemma in a model of Limb Girdle Muscular Dystrophy 2C (LGMD2C). Since recombinant annexin A6 protected against membrane injury, the localization ability of recombinant annexin A6, administered systemically, was assessed in the Sgcg-null mouse model of LGMD2C compared to wildtype controls. Sgcg-null mice lack γ-sarcoglycan, an integral membrane component of the dystrophin glycoprotein complex required for membrane stability and function. Recombinant annexin A6 localized to the muscle membrane of chronically injured, Sgcg-null mice, visualized by strong anti-HIS immunofluorescence signal colocalizing with anti-laminin membrane staining (
This example details the production of recombinant annexin A6 protein (rANXA6) expressed from plasmids in both mammalian cells and prokaryotic cells.
rANXA6 protein production was carried out in a total volume of 4 liters using two mammalian cell lines (Expi293F™ Cells and FreeStyle™ CHO-S Cells, ThermoFisher Scientific). The estimated yield (as determined by Coomassie staining (
The buffers and Immobilized metal affinity chromatography (IMAC) protocols are indicated below in Table 1.
Purification of Annexin 6 (ANXA6) from Expi293F™ Cells. The protein lysate was loaded on to an IMAC column and eluted by 500 mM imidazole. The yield at this stage was determined to be approximately 270 mg. The relevant fractions were pooled and loaded on to an Ion Exchange Q (IExQ) column, and the indicated fractions were pooled. At this stage the yield was approximately 208 mg. These pools were loaded on to a S200 26/60 column in 1×PBS. The final yield was approximately 77 mg. The purity of the recombinant protein was determined to be greater than 95%.
Absolute size exclusion chromatography (aSEC) indicated that the protein was in a monomeric state, and mass spectrometry (MS) showed multiple modifications.
Purification of Annexin 6 (ANXA6) from FreeStyle™ CHO-S Cells. The protein lysate was loaded on to an IMAC column and eluted by 500 mM imidazole. The yield at this point was approximately 78 mg. The relevant fractions were pooled and loaded on to an Ion Exchange Q (IExQ) column, and the indicated fractions were combined in two separate pools. The yield at this stage was approximately 24 mg (approximately 12 mg in each pool). The pools were run on an S200 26/60 column (SEC Run for Pool1 and Pool2). The final yield was determined to be: Pool1=2.25 mg and Pool2=1.9 mg.
Absolute size exclusion chromatography (aSEC) and MS did not show any difference between Pool1 and Pool2, and no difference was detected between the recombinant proteins expressed in Expi293F™ Cells and FreeStyle™ CHO-S Cells.
The rANXA6 protein was also purified from prokaryotic cells. rANXA6 protein production was carried out in a 4 liter low endotoxin purification method in E. coli cells (Rosetta(DE3) competent cells) in TB media. The cells were grown at 30 C for 4 hours and 18 hours. Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer was used at 0.4 mM. The composition of the various buffers used is shown in Table 2. The total yield obtained using the method was 308 mg (approximately 2.5 mg/mL).
The recombinant protein was analyzed by Coomassie staining and immunoblot analysis, as shown in
Purification of Annexin 6 (ANXA6) from E. coli Cells. The protein lysate was loaded on to an IMAC column and eluted by 500 mM imidazole. The yield at this stage was determined to be approximately 546 mg. The relevant fractions were pooled and loaded on to an Ion Exchange Q (IExQ) column, and the indicated fractions were pooled. The yield at this stage was approximately 408 mg. These pools were loaded on to a S200 26/60 column in 1×PBS. After three runs the final yield was approximately 306 mg. The final endotoxin level was approximately 4 EU/mg/10.5 EU/mL. The purity of the recombinant protein was determined to be greater than 95%.
Absolute size exclusion chromatography (aSEC) indicated that the protein was in a monomeric state, and mass spectrometry (MS) showed multiple modifications.
This example details experiments comparing the function of rANXA6 produced in mammalian cells with rANXA6 produced in prokaryotic cells. These experiments were conducted to test whether the activity of an annexin protein (in this case, rANXA6) differed based on whether the annexin protein was produced in mammalian cells versus prokaryotic cells. The predicted posttranslational modification signature for annexin A6 was determined using PTMcode (http://ptmcode.embl.de) and is shown in Table 3. Various forms of posttranslational modification (PTM) identified through mass spectrometry (MS) (e.g., acetylation, nitrosylation, phosphorylation) are indicated in Table 3.
E coli
E coli
E
coli
4
1= identified by mass spectrometry
2= in silico predicted and detected by mass spectrometry
3= in silico predicted but not detected
4= results in this column were obtained using annexin A6 protein obtained from R&D Systems, Inc. (Minneapolis, MN) catalog number 5186-A6-050
5= results in these columns were obtained using annexin A6 protein obtained from a contract research organization (CRO; Evotec SE (Princeton, NJ)) (see Example 5 for general protocols)
As shown in Table 3, the posttranslational modification signature of annexin A6 differs depending on whether the protein is expressed in prokaryotic cells or mammalian cells. It would be expected, therefore, that the altered posttranslational modification signature would result in a change in activity between the annexin A6 protein produced in prokaryotic cells versus mammalian cells. Unexpectedly, however, it was found that the annexin A6 protein produced in prokaryotic cells performed as well as the protein that was produced in mammalian cells in the myofiber injury experiments described below.
Myofiber isolation, laser injury, and dye measurement. For recombinant myofibers studies, myofibers were isolated from wildtype mice. Myofibers were incubated in Ringer's media with 13 μg/ml or 130 μg/ml human recombinant annexin, E coli or HEK cell produced. Bovine serum albumin (BSA) was used as a negative control. FM 4-64 (2.5 μm) was added to the myofibers just prior to imaging. Fibers were subjected to laser-induced damage at room temperature using the Nikon A1R-MP multiphoton microscope. Imaging was performed using a 25×1.1 NA objective directed by the NIS-Elements AR imaging software. FM 4-64 was excited using a 920 nm wavelength laser and emission wavelengths of 575 nm and 629 nm were collected, respectively. To induce laser damage on isolated myofibers, a diffraction limited spot (diameter approximately 410 nm) was created on the lateral membrane of the myofiber using a 920 nm wavelength laser at 10-15% laser power for 1 second. FM 4-64 area was measured using FIJI from a single slice near the middle of the z-stack. Myofiber quality control was based on a number of characteristics including using adherent myofibers with intact sarcomere structure. Myofibers appeared devoid of tears or ruptures induced during the isolation protocol. The region of the myofiber selected for damage was linear and not located on a nucleus or neuromuscular junction.
Statistical analyses were performed with Prism (Graphpad, La Jolla, Calif.). Comparisons relied on ANOVA (1way ANOVA for 1 variable). P-values of less than or equal to 0.05 were considered significant. Error bars represent +/−standard error of the mean (SEM). Results are shown in
These experiments demonstrated that myofibers injured in the presence of extracellular recombinant annexin A6 (rANXA6), produced in E coli or mammalian cells, had similar beneficial effects on enhancing membrane repair and protecting against injury. This was demonstrated across multiple doses, 13 mg/ml and 130 mg/ml, measured as a reduction in FM 4-64 dye uptake after laser injury compared to BSA control treated myofibers.
This example was designed to test the necessity of the amino acid sequence VAAEIL (exon 21 of annexin A6) (SEQ ID NO: 47) in facilitating the localization and organization of the annexin repair cap at the site of membrane injury.
Animals. Wildtype mice from the 129T2/SvEmsJ background were bred and housed in a specific pathogen free facility on a 12-hour light/dark cycle and fed ad libitum in accordance with the Northwestern University's Institutional Animal Care and Use Committee regulations. 129T2/SvEmsJ (129T2) mice were purchased from the Jackson Laboratory (Ben Harbor, Me.; Stock #002065). Two to three-month-old male and female were used for all wildtype mouse experiments.
Plasmids. Plasmids encoding annexin A6 with a carboxyl-terminal turboGFP tag were obtained from Origene (Rockville, Md.). Subcloning of annexin A6 to replace the GFP tag with tdTomato (Addgene) was performed by Mutagenix (Suwanee, Ga.). Site directed mutagenesis was performed by Mutagenix on annexin A6-GFP to generate the construct lacking the VAAEIL (SEQ ID NO: 47) sequence. Plasmid DNA was isolated using the Qiagen endo-free Maxi prep kit (Qiagen #12362).
Electroporation, myofiber isolation, laser injury, cap and vesicle measurement. Flexor digitorum brevis (FDB) fibers were transfected with endo-free plasmid DNA by in vivo electroporation. Methods were described previously in Demonbreun et al JoVE 2015. Briefly, the hindlimb footpad was injected with 10 μl of hyaluronidase (8 units) (H4272, Sigma, St. Louis, Mo.). Two hours post injection up to 20 μl of 2 μg/μl endotoxin free plasmid was injected into the footpad. Electroporation was conducted by applying 20 pulses, 20 ms in duration/each, at 1 Hz, at 100 V/cm. Animals were allowed to recover for a minimum of seven days and not more than ten days after electroporation to avoid examining injured muscle and to allow sufficient time for plasmid expression.
FDB muscle was removed and individual myofibers were isolated and imaged (Demonbreun et al., JoVE, 2015). Fibers were dissected and laser damaged as described (Demonbreun et al., JoVE 2015; Swaggart et al., PNAS 2014). Briefly, fibers were dissociated in 0.2% BSA plus collagenase type II (Cat #17101, Life Technologies, Grand Island, N.Y.) for up to 90 minutes with intermittent trituration at 37 degrees in 10% CO2. Fibers were then moved to Ringers solution and placed on MatTek confocal microscopy dishes (Cat #P35G-1.5-14-C, MatTek, Ashland Mass.). After one hour, fibers were adhered and the Ringers was replaced with 1 ml of fresh Ringers solution. Imaging and ablation was performed on the Nikon A1R laser scanning confocal equipped with GaSP detectors through a 60× Apo lambda 1.4 NA objective driven by Nikon Elements AR software. A single pixel set as 120 nm (0.0144 μm2) was ablated using the 405 nm laser at 100% power for up to 5 seconds. Z-stack projections were acquired from consecutive acquisitions after the final time-lapse frame, approximately 4 minutes post damage, with a 0.125 μM step size between slices. Z-stack renderings were constructed in FIJI. Fibers expressing similar levels of tagged protein were compared.
It was then questioned whether the presence of the VAAEIL (SEQ ID NO: 47) sequence in annexin A6 influenced annexin A6 localization upon muscle membrane injury. Myofibers were electroporated with full-length annexin A6-tdTomato and A6-GFP lacking VAAEIL (SEQ ID NO: 47) and subsequently laser-injured. Annexin A6 isoforms with and without the VAAEIL (SEQ ID NO: 47) sequence localized to the site of muscle membrane injury forming a repair cap (
This application claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62/783,619, filed Dec. 21, 2018 and U.S. Provisional Patent Application No. 62/903,525, filed Sep. 20, 2019, which are incorporated herein by reference in their entirety.
This invention was made with government support under grant numbers U54 AR052646 and R01 NS047726 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/68168 | 12/20/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62903525 | Sep 2019 | US | |
| 62783619 | Dec 2018 | US |
