Home ❯ Research ❯ Funded Studies
Current Grant
07/24 – 06/25
The major focus of our work in the coming year will be to analyze the potential benefits of our experimental gene therapy on dysferlin-null mice. As we reported last year, we have identified a highly specific, effective, and efficient fragment of dysferlin to introduce into muscle by AAV. We will be assessing its effectiveness in restoring the normal physiology and preventing the histopathology that develops over time in dysferlin-null muscles. Our hope is that, if it works, it will provide a way of slowing or halting the progression in patients with LGMD R2.
Our studies of pathogenic point mutants of the C2 domains of dysferlin are now almost complete, but we have a number of studies of the transmembrane domain ongoing, with the goal of learning if this domain is needed for activity and for dimer formation. Similarly, our studies of myoferlin and its relationship to dysferlin are ongoing, with the expectation that they will be completed this year.
Previous Grant Period
07/23 – 06/24
Our work over the past year focused on making progress on our ongoing projects to replace dysferlin with small but fully active pieces of the protein, with the goal of developing a gene therapy for LGMD R2, completing our studies of point mutants in the C2 domains of the protein, and understanding the role of dysferlin’s transmembrane domain. We also initiated a new projects to understand the possible relationship between dysferlin and myoferlin.
Our development of a potential gene therapy has focused on identifying the most active fragment of dysferlin that, even expressed at very low concentrations, would replace wild type dysferlin and fully support the normal mechanisms of calcium release in otherwise dysferlin-null muscles. We have found that the C2A domain of dysferlin, targeted by other C2 domains to the triad junction, are highly specific and effective. We have now introduced them into adeno-associated virus (AAV) and transduced dysferlin-null mice, to learn if this gene therapy will restore the normal physiology of dysferlin-null muscle.
Our studies of point mutants have shown that pathogenic mutants in every C2 domain of dysferlin except C2B also cause disruption of normal calcium signaling. This suggests that each domain except C2B is essential for ensuring that calcium is only released to activate contraction when the muscle is electrically stimulated – a key feature of healthy muscle. Surprisingly, however, changing the transmembrane domain of dysferlin drastically had no effect on its function.
Our studies of myoferlin suggest that, like dysferlin, it can control the mechanisms of calcium release in skeletal muscle, but that it does so less efficiently than dysferlin, probably because it is not expressed as highly and does not target the sites controlling calcium release as effectively. They further indicate that dysferlin and myoferlin interact in muscle fibers, which is consistent with earlier findings that dysferlin can interact with itself to form a dimer of two dysferlin molecules. This suggests that enhancing myoferlin’s expression in muscle with low levels of dysferlin could provide clinical benefit.
Previous Grant Period
05/22 – 04/23
We spent much of our grant year optimizing the C2A domain constructs for use in our adeno-associated virus (AAV) experiments. We had previously found that placing the C2A domain of dysferlin with the adjacent Romeo epitope in tandem with C2 domains from another muscle protein allowed our newly created hybrid protein to concentrate at the triad junctions of dysferlin-null skeletal muscle, where dysferlin would normally be concentrated. This protein had the two key activities of native dysferlin that we have been studying: proper regulation of Ca2+ signaling, and good support of membrane repair. The problem we faced was with the controls, for which we had originally planned to use inactive variants of the C2A domain, such as the V67D or the A84R mutants. When placed next to the same muscle protein C2 domains, neither was expressed well in dysferlin-null muscle – not nearly as well as the wild type. As the amount of the virally expressed control protein should be approximately the same as our experimental one containing C2A and Romeo, we decided to use virus expressing the other C2 domains alone. An added advantage of this approach is that this hybrid protein also concentrates at the triad junctions but it is inactive both in stabilizing Ca2+ signaling and in promoting membrane repair (the latter was assayed by Noah Weisleder and his colleagues at OSU in Columbus, OH). We can now express the experimental and control proteins as mCrimson fusion proteins and have provided the plasmid constructs to a commercial facility for production of the AAV.
In a separate series of experiments, we began to investigate the relationship between the abnormal Ca2+ signaling that occurs in dysferlin-null muscle after a mild injury, which is mediated by a process call Ca2+-induced Ca2+ release, or CICR, and the normal signaling seen in healthy muscle, even after injury, which is mediated by what is called voltage-induced Ca2+ release, or VICR. CICR is a phenomenon that underlies many diseases of muscle and it is generally thought that suppressing it, when it occurs, can improve muscle health. Our results so far suggest that VICR and CICR may be interconvertible and that their interconversion may be mediated by enzymes that add or remove phosphate residues from a protein or proteins at the triad junction. We are trying to identify these enzymes now, as drugs that affect their activity may be useful in suppressing some of the abnormal Ca2+ signaling that is typical of dysferlinopathic muscle and thereby improve the length of time over which dysferlinopathic muscle can remain functional. We are currently seeking separate funding for this aspect of our research.
Previous Grant Period
05/21 – 04/22
Our most recent experiments have identified a construct containing the C2A domain and the nearby Romeo epitope of dysferlin, linked to C2 domains from another protein, that like dysferlin itself is active in protecting dysferlin-null A/J myofibers from the effects of hypoosmotic shock injury. It is also active in assays of sarcolemmal wounding, carried out in the Weisleder laboratory. Activity is seen even at low levels of expression of the chimeric construct. We are now moving this construct into a vector that, after insertion into AAV, should allow us to visualize its expression in murine muscle and to determine if it reverses the known phenotypes associated with dysferlinopathy in vivo. As a control, we will be using the same construct but with the C2A domain carrying the V67D mutation, which is inactivating but which is still expressed in muscle.
We have also been examining the role of Ca2+ in the myoplasm and at the triad junction in promoting Ca2+-induced Ca2+ release (CICR), induced in dysferlin-null A/J muscle by hypoosmotic shock. We find that BAPTA is more efficient than Fluo-4, Rhod-2 or EGTA in rescuing the A/J fibers and converting them to the wild type phenotype, in which CICR is normally suppressed. BAPTA has a higher affinity for Ca2+ than Fluo-4 and Rhod-2 and binds Ca2+ much more rapidly than EGTA. Placing a Ca2+ binding moiety (a variant of GCaMP) with high affinity and rapid binding kinetics at the N-terminus of dysferlin missing its C2A domain is also sufficient to convert the A/J phenotype to wild type. This construct targets the triad junction specifically. Our results therefore suggest that rapid, high affinity binding of Ca2+ in the triad junction is sufficient to suppress CICR and restore the wild-type phenotype to dysferlinopathic muscle. It further suggests that a key role of dysferlin’s C2A domain is to bind Ca2+ at the triad junction.
Previous Grant Period
02/20 – 06/21
In the thirteenth year of support from the Jain Foundation, we focused on refining our understanding of the role of the C2A domain of dysferlin in regulating Ca2+ signaling in myofibers before and after a mild injury. Our previous results indicated that the C2A domain has a unique role in sustaining Ca2+ signaling after mild hypoosmotic shock. To assess its specificity, we introduced inactivating point mutations as well as polymorphisms, substituted other, homologous C2 domains for C2A, and examined the alternatively spliced variant, C2Av1. The variant and polymorphic mutations sustained normal activity. Other constructs failed to do so. We found, however, that the C2 domain of PKCα targets the triad junction regions, where dysferlin normally concentrates. We therefore created constructs of C2A and C2PKCα and showed that these are more active at lower levels of expression than any other constructs we have studied. Additional evidence suggested that C2PKCα-C2A was likely to function because it concentrates at triad junctions, where it can bind Ca2+ effectively.
In an extension of our ongoing characterization of the C2 domains of dysferlin, we also completed our studies of point mutations that are thought to inactivate the protein’s role in vivo. Our results indicate that nearly all of them also inactivate dysferlin’s role in regulating Ca2+ signaling. This supports our earlier conclusion that all the C2 domains of dysferlin, with the possible exception of C2B, are required for normal Ca2+ homeostasis.
Previous Grant Period
02/12 – 01/20
Current Grant
07/24 – 06/25
The major focus of our work in the coming year will be to analyze the potential benefits of our experimental gene therapy on dysferlin-null mice. As we reported last year, we have identified a highly specific, effective, and efficient fragment of dysferlin to introduce into muscle by AAV. We will be assessing its effectiveness in restoring the normal physiology and preventing the histopathology that develops over time in dysferlin-null muscles. Our hope is that, if it works, it will provide a way of slowing or halting the progression in patients with LGMD R2.
Our studies of pathogenic point mutants of the C2 domains of dysferlin are now almost complete, but we have a number of studies of the transmembrane domain ongoing, with the goal of learning if this domain is needed for activity and for dimer formation. Similarly, our studies of myoferlin and its relationship to dysferlin are ongoing, with the expectation that they will be completed this year.
Previous Grant Period
07/23 – 06/24
Our work over the past year focused on making progress on our ongoing projects to replace dysferlin with small but fully active pieces of the protein, with the goal of developing a gene therapy for LGMD R2, completing our studies of point mutants in the C2 domains of the protein, and understanding the role of dysferlin’s transmembrane domain. We also initiated a new projects to understand the possible relationship between dysferlin and myoferlin.
Our development of a potential gene therapy has focused on identifying the most active fragment of dysferlin that, even expressed at very low concentrations, would replace wild type dysferlin and fully support the normal mechanisms of calcium release in otherwise dysferlin-null muscles. We have found that the C2A domain of dysferlin, targeted by other C2 domains to the triad junction, are highly specific and effective. We have now introduced them into adeno-associated virus (AAV) and transduced dysferlin-null mice, to learn if this gene therapy will restore the normal physiology of dysferlin-null muscle.
Our studies of point mutants have shown that pathogenic mutants in every C2 domain of dysferlin except C2B also cause disruption of normal calcium signaling. This suggests that each domain except C2B is essential for ensuring that calcium is only released to activate contraction when the muscle is electrically stimulated – a key feature of healthy muscle. Surprisingly, however, changing the transmembrane domain of dysferlin drastically had no effect on its function.
Our studies of myoferlin suggest that, like dysferlin, it can control the mechanisms of calcium release in skeletal muscle, but that it does so less efficiently than dysferlin, probably because it is not expressed as highly and does not target the sites controlling calcium release as effectively. They further indicate that dysferlin and myoferlin interact in muscle fibers, which is consistent with earlier findings that dysferlin can interact with itself to form a dimer of two dysferlin molecules. This suggests that enhancing myoferlin’s expression in muscle with low levels of dysferlin could provide clinical benefit.
Previous Grant Period
05/22 – 04/23
We spent much of our grant year optimizing the C2A domain constructs for use in our adeno-associated virus (AAV) experiments. We had previously found that placing the C2A domain of dysferlin with the adjacent Romeo epitope in tandem with C2 domains from another muscle protein allowed our newly created hybrid protein to concentrate at the triad junctions of dysferlin-null skeletal muscle, where dysferlin would normally be concentrated. This protein had the two key activities of native dysferlin that we have been studying: proper regulation of Ca2+ signaling, and good support of membrane repair. The problem we faced was with the controls, for which we had originally planned to use inactive variants of the C2A domain, such as the V67D or the A84R mutants. When placed next to the same muscle protein C2 domains, neither was expressed well in dysferlin-null muscle – not nearly as well as the wild type. As the amount of the virally expressed control protein should be approximately the same as our experimental one containing C2A and Romeo, we decided to use virus expressing the other C2 domains alone. An added advantage of this approach is that this hybrid protein also concentrates at the triad junctions but it is inactive both in stabilizing Ca2+ signaling and in promoting membrane repair (the latter was assayed by Noah Weisleder and his colleagues at OSU in Columbus, OH). We can now express the experimental and control proteins as mCrimson fusion proteins and have provided the plasmid constructs to a commercial facility for production of the AAV.
In a separate series of experiments, we began to investigate the relationship between the abnormal Ca2+ signaling that occurs in dysferlin-null muscle after a mild injury, which is mediated by a process call Ca2+-induced Ca2+ release, or CICR, and the normal signaling seen in healthy muscle, even after injury, which is mediated by what is called voltage-induced Ca2+ release, or VICR. CICR is a phenomenon that underlies many diseases of muscle and it is generally thought that suppressing it, when it occurs, can improve muscle health. Our results so far suggest that VICR and CICR may be interconvertible and that their interconversion may be mediated by enzymes that add or remove phosphate residues from a protein or proteins at the triad junction. We are trying to identify these enzymes now, as drugs that affect their activity may be useful in suppressing some of the abnormal Ca2+ signaling that is typical of dysferlinopathic muscle and thereby improve the length of time over which dysferlinopathic muscle can remain functional. We are currently seeking separate funding for this aspect of our research.
Previous Grant Period
05/21 – 04/22
Our most recent experiments have identified a construct containing the C2A domain and the nearby Romeo epitope of dysferlin, linked to C2 domains from another protein, that like dysferlin itself is active in protecting dysferlin-null A/J myofibers from the effects of hypoosmotic shock injury. It is also active in assays of sarcolemmal wounding, carried out in the Weisleder laboratory. Activity is seen even at low levels of expression of the chimeric construct. We are now moving this construct into a vector that, after insertion into AAV, should allow us to visualize its expression in murine muscle and to determine if it reverses the known phenotypes associated with dysferlinopathy in vivo. As a control, we will be using the same construct but with the C2A domain carrying the V67D mutation, which is inactivating but which is still expressed in muscle.
We have also been examining the role of Ca2+ in the myoplasm and at the triad junction in promoting Ca2+-induced Ca2+ release (CICR), induced in dysferlin-null A/J muscle by hypoosmotic shock. We find that BAPTA is more efficient than Fluo-4, Rhod-2 or EGTA in rescuing the A/J fibers and converting them to the wild type phenotype, in which CICR is normally suppressed. BAPTA has a higher affinity for Ca2+ than Fluo-4 and Rhod-2 and binds Ca2+ much more rapidly than EGTA. Placing a Ca2+ binding moiety (a variant of GCaMP) with high affinity and rapid binding kinetics at the N-terminus of dysferlin missing its C2A domain is also sufficient to convert the A/J phenotype to wild type. This construct targets the triad junction specifically. Our results therefore suggest that rapid, high affinity binding of Ca2+ in the triad junction is sufficient to suppress CICR and restore the wild-type phenotype to dysferlinopathic muscle. It further suggests that a key role of dysferlin’s C2A domain is to bind Ca2+ at the triad junction.
Previous Grant Period
02/20 – 06/21
In the thirteenth year of support from the Jain Foundation, we focused on refining our understanding of the role of the C2A domain of dysferlin in regulating Ca2+ signaling in myofibers before and after a mild injury. Our previous results indicated that the C2A domain has a unique role in sustaining Ca2+ signaling after mild hypoosmotic shock. To assess its specificity, we introduced inactivating point mutations as well as polymorphisms, substituted other, homologous C2 domains for C2A, and examined the alternatively spliced variant, C2Av1. The variant and polymorphic mutations sustained normal activity. Other constructs failed to do so. We found, however, that the C2 domain of PKCα targets the triad junction regions, where dysferlin normally concentrates. We therefore created constructs of C2A and C2PKCα and showed that these are more active at lower levels of expression than any other constructs we have studied. Additional evidence suggested that C2PKCα-C2A was likely to function because it concentrates at triad junctions, where it can bind Ca2+ effectively.
In an extension of our ongoing characterization of the C2 domains of dysferlin, we also completed our studies of point mutations that are thought to inactivate the protein’s role in vivo. Our results indicate that nearly all of them also inactivate dysferlin’s role in regulating Ca2+ signaling. This supports our earlier conclusion that all the C2 domains of dysferlin, with the possible exception of C2B, are required for normal Ca2+ homeostasis.
Previous Grant Period
02/12 – 01/20
