Prothelia - Pipeline

Prothelia Pipeline

	Discovery	Optimization	Preclinical	Phase I	Phase II

PRT-20

LAM-111

PRT-300

Disease Focus

Prothelia is dedicated to serving patients with muscular dystrophy

Duchenne muscular dystrophy (DMD)
Congenital muscular dystrophy type 1A (MDC1A)
Dystrophin-deficient and X-linked dilated cardiomyopathy (XLDCM)
Limb girdle muscular dystrophy type 2B (LGMD2B)
Congenital muscular dystrophy type 1D (MDC1D)

Prothelia has exclusive worldwide license to the intravenous protein therapy LAM-111, and the small molecules PRT-20 and PRT-300.

LAM-111, PRT-20 and PRT-300 will treat ALL patients with Duchenne muscular dystrophy (DMD) and Congenital muscular dystrophy type 1A (MDC1A), regardless of their respective mutations.

LAM-111, PRT-20 and PRT-300 will enhance the effectiveness of other therapies in development for DMD such as utrophin enhancement (BioMarin) and the exon skipping therapies (AVI BioPharma and Prosensa).

RECOMBINANT HUMAN LAMININ-111 FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY

Bradley L. Hodges PhD, Prothelia Inc. 30 Haven Street, Milford MA 01757, bradhodges@prothelia.com

SUMMARY. Defective adhesion between the interior and exterior of skeletal and cardiac myofibers is a common etiological event in many forms of muscular dystrophy, which may arise from defects in cytoskeletal proteins such as dystrophin and plectin, membrane proteins such as the sarcoglycans and alpha dystroglycan, or extracellular matrix proteins such as laminin-211 and collagen VI (1-15). Duchenne muscular dystrophy (DMD) affects 1 in 3500 male births and is caused by a deficiency of dystrophin, a large multifunctional cytoskeletal protein that maintains the sarcolemmal integrity of skeletal and cardiac muscles (1-11). The loss of dystrophin in DMD leads to the secondary loss of alpha dystroglycan; the predominant laminin receptor found on the membranes of skeletal and cardiac myofibers, resulting in a substantial loss in laminin-dependent adhesion (Figure 1A). The sarcolemma (membrane) of DMD patients is thus abnormally sensitive to contraction-induced damage, eliciting cycles of muscle degeneration and regeneration which eventually become exhausted (3-15). As DMD progresses patients lose the ability to walk, feed and breathe without assistance, the quality of life becomes extremely poor and patients usually die within their third decade of life from respiratory infection or cardiac failure (2). A single systemic dose of LAM-111 efficiently distributed to skeletal and cardiac muscles of mdx mouse model of DMD, resulted in an enhancement of alpha7 integrin, blunted myofiber degeneration, normalized serum creatine kinase and provided a potent and durable protection of mdx muscles against the damaging effects of eccentric treadmill exercise (34). Thus, the body of evidence suggests that LAM-111 based enhancement of alpha7 integrin represents a powerful approach to treat DMD and MDC1A and may treat other forms of muscular dystrophy in which sarcolemma integrity is compromised. We are developing recombinant human laminin-111 (rhLAM-111) for the treatment of Duchenne muscular dystrophy (DMD), and we have secured a corporate partner to assist in the manufacture, preclinical and clinical development and commercialization of rhLAM-111.

LAM-111 can be systemically delivered to target both skeletal and cardiac myofibers
LAM-111 binds with high affinity to the two predominant laminin receptors found on skeletal and cardiac muscle; alpha dystroglycan and the alpha7beta1 integrin
LAM-111 induces the transcription of the alpha7 integrin subunit and increases the sarcolemmal localization the alpha7beta1 integrin and utrophin, two molecules previously shown to ameliorate the progression of disease in mouse models of DMD
The alpha7 integrin is expressed in all muscular dystrophy patients, and enhancement of alpha7beta1 integrin via LAM-111 should benefit all DMD patients regardless of their respective dystrophin mutations
LAM-111 targets cardiac myofibers and may also mitigate the cardiac manifestations of DMD
All DMD patients express LAM-111 suggesting that a detrimental immune response to recombinant human LAM-111 is unlikely

FIGURE 1: STABILIZATION OF DYSTROPHIN DEFICIENT MUSCLES WITH rhLAM-111
APPROACHES TO TREATMENT OF DMD
ENHANCEMENT OF THE ALPHA7 INTEGRIN AS A TREATMENT FOR DMD
LAM-111 UPREGULATES ALPHA7 INTEGRIN AND UTROPHIN
APPROACHES UNDER DEVELOPMENT FOR TREATMENT OF DMD
POTENTIAL FOR IMMUNE RESPONSE TO rhLAM-111
REFERENCES

FIGURE 1: STABILIZATION OF DYSTROPHIN DEFICIENT MUSCLES WITH rhLAM-111

Figure 1. Alpha dystroglycan (αD) and the alpha7beta1 (α7β1) integrin are the dominant laminin receptors on skeletal and cardiac muscle. Dystrophin deficiency in DMD and mdx mice results in a severe secondary deficiency of alpha dystroglycan and as a result dystrophin deficient muscles are sensitive to damage and degeneration. In the absence of dystrophin, the alpha7beta1 integrin becomes the predominant junctional and extrajunctional LAM-211/221 receptor in DMD/mdx skeletal muscles (A). Intravenous delivery of LAM-111 onto DMD/mdx skeletal muscles (in concert with existing LAM-211) is hypothesized to stabilize myofibers through recruitment of existing alpha7beta1 integrin and induction of further alpha7 integrin expression (B).

APPROACHES TO TREATMENT OF DMD. As DMD progresses patients lose the ability to walk, feed and breathe without assistance, the quality of life becomes extremely poor and patients usually die within their third decade of life from respiratory infection or cardiac failure (2). There are no approved therapies for DMD and the only available treatments are steroids, beta blockers and ACE inhibitors (16-17). To prevent sarcolemmal instability and the ensuing muscle degeneration in DMD, any intervention for DMD must restore the transmembrane linkage between the interior contractile apparatus, muscle membrane and exterior basal lamina (1-15), and in skeletal and cardiac muscle alpha7beta1 integrin and alpha dystroglycan are the dominant laminin receptors that maintain these transmebrane linkages (1-15, 18-27).

ENHANCEMENT OF THE ALPHA7 INTEGRIN AS A TREATMENT FOR DMD. The alpha7 integrin subunit is a genetic modifier of muscular dystrophy; double knockout (dKO) mice of alpha7/dystrophin, alpha7/gamma sarcoglycan or alpha7/alpha dystroglycan present with a more severe muscular dystrophy than that of individual knockout mice, and the reduction of alpha7 integrin that occurs secondary to merosin deficiency contributes to the progression of muscle pathology in dyW mice (21, 22, 27, 28-33). Severely dystrophic (mdx/utro-/- dKO) mice that overexpress 2-3X the normal amount of the alpha7X2B integrin, the predominant integrin isoform in skeletal muscle, exhibited increased weight, muscle regenerative capacity and lifespan, as well as reduced kyphosis, joint contractures, cardiomyopathy, central nuclei and Evans blue dye uptake (18, 19).

LAM-111 UPREGULATES ALPHA7 INTEGRIN AND UTROPHIN. Approximately 90% of the therapeutic effect of LAM-111 is through enhancement and engagement of the alpha7beta1 integrin, while the remaining therapeutic effect is through utrophin enhancement (34). The alpha7 integrin alternatively spliced X1 and X2 extracellular domains dictate the affinity of alpha7beta1 integrin to various isoforms of laminin, while the alternatively spliced A and B cytoplasmic domains dictate the localization of alpha7 along the myofiber (10, 35-40). The alpha7X2Bbeta1 integrin isoform predominates in skeletal muscle and possesses a ~3-5X higher affinity for LAM-111 versus LAM-211, and the affinity of alpha dystroglycan for LAM-111 is marginally less than its affinity for LAM-211 (10, 35-43). LAM-111 is not expressed in normal or dystrophic muscle, is the most closely related to LAM-211 (43-50), and transgenic expression of laminin alpha1 chain (LAM-111) in the dyW mouse model of MCD1A alleviated the myopathic phenotype and restored the alpha7 integrin to the muscle surface (51). DMD and mdx myotubes adhere to LAM-111 to a far greater extent than to LAM-211/221, and this binding is mediated by the alpha7beta1 integrin (52). LAM-111 restores defective muscle regeneration in the alpha7 integrin KO mouse, and treatment of human and mouse muscle cells in vitro with murine LAM-111 (derived from EHS sarcoma) enhanced the expression of the alpha7 integrin mRNA and protein (34, 53). A single systemic dose of LAM-111 efficiently distributed to skeletal and cardiac muscles of mdx mice, resulted in an enhancement of alpha7 integrin, blunted myofiber degeneration, normalized serum creatine kinase and provided a potent and durable protection of mdx muscles against the damaging effects of eccentric treadmill exercise (34). Characterization of mdx/utro -/- dKO mouse expressing transgenic (rat) alpha7X2B integrin demonstrated that the 150% increase in alpha7X2B could fully account for the amelioration of disease (18, 19), and is consistent with the presented data demonstrating that the enhanced expression of alpha7 integrin in LAM-111 treated mdx mice likely accounts for the observed therapeutic effect (18, 19, 34). Thus, the body of evidence suggests that LAM-111 based enhancement of alpha7 integrin represents a powerful approach to treat DMD and MDC1A and may treat other forms of muscular dystrophy in which sarcolemma integrity is compromised. Alpha7 integrin deficiency is a partial embryonic lethal mutation in mice and extensive interrogation of patient samples manifesting idiosyncratic myopathies has found only three patients worldwide possessing defects in the alpha7 integrin (54-56). Therefore, the alpha7 integrin should be expressed in all patients with muscular dystrophy and superphysiologic enhancement of the alpha7 integrin is not expected to be toxic (18, 19), nor immunogenic.

APPROACHES UNDER DEVELOPMENT FOR TREATMENT OF DMD. Gene and stem cell therapy, mRNA rescue technologies such as Ataluren and exon skipping, small molecule enhancement of the so-called "booster genes" such as utrophin, alpha7 integrin and GALGT, and biologics such as biglycan and laminin-111 (LAM-111) are under development for treatment of DMD (12, 61-68). We anticipate that LAM-111 will follow a development path that is similar to Myozyme®, an intravenous protein therapy for treatment of Pompe disease, and we anticipate that rhLAM-111 should have a tremendous impact on the life and welfare of patients with DMD. RhLAM-111 should also benefit patients with other forms of muscular dystrophy such as MDC1A, MDC1D and LGMD2B in which upregulation of the alpha7 integrin is therapeutic.

POTENTIAL FOR IMMUNE RESPONSE TO RHLAM-111. There are many factors that contribute to the development of an immune response to a given therapeutic protein: structural features such as 1) sequence variation or 2) glycosylation profile, 3) subcutaneous or intramuscular routes of administration tend to be more immunogenic than the intravenous route, 4) genetic characteristics of patients, 5) the extent to which the patient synthesizes the sequence of the therapeutic peptide, 6) improper formulation or handling, 7) improper storage conditions that lead to denatured, oxidized aggregates, 8) contaminants or impurities in the preparation, and 9) dose and length of treatment (57). Importantly, the consequence of an immune response to an intravenous protein therapy is not equivalent to that directed against a cell or gene therapy; if an antibody titer develops against a therapeutic protein, the therapeutic protein can often be readministered (57-59). In such cases the patient may receive prophylactic acetaminophen, diphenhydramine and dexamethasone, more therapeutic protein may need to be administered, the pharmacokinetics of the given therapeutic may become modified, and except for a few documented cases (60), the patients continue to respond to the given therapeutic protein. All humans produce LAM-111 (50), and aside from those patients who possess silent mutations in their laminin alpha 1 chain, they should be tolerant to the peptide sequence of rhLAM-111. Recombinant hLAM-111 will have a mammalian-like glycosylation profile, will be delivered intravenously and therefore we will have minimized all sources of antigenicity. Only post-marketing surveillance will determine if other post-production sources of antigenicity (factors 4 through 9 mentioned above) lead to the development of severe adverse events.

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TREATMENT OF DYSTROPHIN DEFICIENT DILATED CARDIOMYOPATHY (DMD, XLDCM) WITH LAMININ-111

Bradley L. Hodges PhD, Prothelia Inc. 30 Haven Street, Milford MA 01757, bradhodges@prothelia.com

SUMMARY. Dystrophin deficiency (DMD), predisposing factors, or environmental insults can induce a pathologic form of decompensated cardiac hypertrophy characterized by increased myocardial mass and wall thickness, reduced ventricular volume and reduced systolic and diastolic function. Left untreated decompensated hypertrophy can deteriorate into dilated cardiomyopathy (DCM) characterized by ventricular wall thinning, increased ventricular volume, reduced systolic and diastolic function, fibrosis, myocyte death and heart failure. We are evaluating whether laminin-111 will mitigate the development of dilated cardiomyopathy in DMD.

FIGURE 1: CELLS AND STRUCTURAL FEATURES OF CARDIOMYOCYTES
DILATED CARDIOMYOPATHY (DCM)
MECHANOTRANSDUCTION, INTEGRINS, AND CARDIAC HYPERTROPHY
DYSTROPHIN-DEFICIENT DILATED CARDIOMYOPATHY
THE BASAL LAMINA OF NORMAL AND DYSTROPHIC MYOCARDIUM
LAM-111: AN INTRAVENOUS PROTEIN THERAPY FOR DYSTROPHIN-DEFICIENT DCM
LONG-TERM IMPLICATIONS OF BETA1 INTEGRIN ENGAGEMENT
REFERENCES

FIGURE 1: CELLS AND STRUCTURAL FEATURES OF CARDIOMYOCYTES

Figure 1. Two neighboring cardiomyocytes are shown with attending interstitial fibroblasts and a capillary embedded within the extracellular matrix. The alpha7beta1 integrin is the predominant laminin binding receptor in DMD cardiac myocytes and is localized at regions of contact (the costameres and intercalated discs) shown in red. Laminin-111 is expected to strengthen these contacts and mitigate the development of dilated cardiomyopathy in DMD.

DILATED CARDIOMYOPATHY (DCM). The myocardium is able to undergo compensatory hypertrophic changes as a normal physiologic response to increase workloads such as those following chronic exercise or pregnancy. Through the collusion of various growth factors, cytokines, neurohumoral inputs, and mechanosensory receptors the cardiomyocytes and surrounding interstitial cells can "sense" the increased workload, and a series of intracellular signaling responses is then initiated that ultimately leads to increased myofibrillar contractile mass and a compensatory cardiomyocyte hypertrophy (1). A return to a sedentary or post-partum state induces a reversion of hypertrophic cardiomyocytes to preload dimensions. However, certain genetic deficiencies, predisposing factors, or environmental insults can induce a pathologic form of decompensated hypertrophy characterized by increased myocardial mass and wall thickness, reduced ventricular volume and reduced systolic and diastolic function. Left untreated decompensated hypertrophy can deteriorate into dilated cardiomyopathy (DCM) characterized by ventricular wall thinning, increased ventricular volume, reduced systolic and diastolic function, fibrosis, myocyte death and heart failure (1).

Molecules of the cell cytoskeleton, the membrane and extracellular matrix work cooperatively to transmit the force of myocardial contraction along the lateral and longitudinal axis of individual cardiomyocytes and fibroblasts, and globally throughout the entire myocardium (2, 3). In cardiac myofibers the predominant transmembrane receptor systems responsible for bridging the contractile apparatus to the extracellular matrix are dystrophin and the dystrophin associated proteins and the integrins (4, 5). Dystrophin and the dystrophin-associated proteins are localized at costameric and extracostameric locations of the lateral sarcolemma, but are absent from the intercalated discs (6, 7). The integrins (predominantly alpha7Bbeta1) are localized at both costameres and the intercalated discs (5, 6) (Figure 1). The cadherins make homotypic associations between neighboring cardiomyocytes and are localized at the intercalated discs and desmosomes (8, 9, 10). Thus, dystrophin maintains sarcolemma integrity along the lateral axis of the cardiomyocyte, the cadherins do so along the longitudinal axis, and integrins maintain the sarcolemma integrity at both lateral and longitudinal locations (Figure 1). This is a simplified view of muscle architecture as many other cytoskeletal and membrane components, cytoplasmic signaling molecules and various components of the extracellular matrix integrate with dystrophin, the integrins and cadherins to maintain a properly functioning contractile unit, and a deficiency in one of many different attachment proteins is the cause of various muscular dystrophies and/or cardiac myopathies (8, 10, 11).

MECHANOTRANSDUCTION, INTEGRINS, AND CARDIAC HYPERTROPHY. Integrins are a large family of transmembrane heterodimeric proteins composed of one of eighteen alpha subunits and one of eight beta subunits that in various combinations bind laminin, fibronectin, and collagen, among other molecules of the basal lamina (12). Beta1-containing integrins are the primary mechanosensory inputs that communicate stretch signals in the myocardium (4, 5, 9, 13-16), and a complex of adapter proteins that link the beta1 integrin cytoplasmic domain to the cytoskeleton also mediate the action of second messengers that communicate stretch signals to the cell interior (17, 18).

The alpha7beta1 integrin is the predominant laminin binding integrin expressed on skeletal and cardiac myofibers and is localized at all major points of cardiomyocyte adhesion (Figure 1). The alpha7 integrin exists as four predominant isoforms; one of two alternatively spliced extracellular X1 or X2 domains are joined with one of two alternatively spliced cytoplasmic A and B domains (2). The alpha7A cytoplasmic domain is expressed only in skeletal muscle and in adult cardiomyocytes the alpha7X1B and alpha7X2B isoforms heterodimerize exclusively with the beta1D integrin isoform to form receptors for laminin-211 (LAM-211) (2, 4, 5, 19-21). Beta1A-containing integrins are found predominantly on myocardial endothelium and fibroblasts. The beta1A integrin heterodimerizes with the alpha3 and alpha6 integrin subunits to form laminin receptors, and beta1A heterodimerizes with the alpha5 integrin to form a fibronectin receptor. (19, 20, 22).

Beta1 integrin deficiency sensitizes cardiomyocytes to the detrimental effects of pressure overload (13-16). Mice possessing a cardioventricular-specific knockdown of beta1 integrin develop a dilated cardiomyopathy, fibrosis, intolerance to pressure overload (POL) and reduced survival following transverse aortic constriction (TAC) (13). Similarly, reduction of wildtype beta1 integrin following cardiac-specific expression of a transgenic dominant-negative beta1 integrin results in a dilated hypertrophy, depressed contractility and relaxation, depressed ventricular pressure and heart rate, and a ~400% elevation in ANF and beta-myosin heavy chain (14). Similar transgenic lines that expressed greater levels of the dominant-negative beta1 integrin exhibited diffuse fibrotic changes of the heart and died around the time of birth (14). Deficiencies of molecules that bind the beta1 integrin cytoplasmic domain also sensitize cardiomyocytes to pressure overload. Melusin is a calcium-binding scaffold protein that interacts specifically with the beta1 integrin cytoplasmic domain, is expressed in skeletal and cardiac muscle, and is an important mediator of mechanosensory induced signals emanating from the beta 1 integrin (23). Pressure overload of melusin KO mice results in reduced left ventricular hypertrophy and favors the transition to DCM despite normal expression and localization of the alpha7Bbeta1D integrin (24). Conversely, transgenic overexpression of melusin prevents left ventricular dilation, apoptosis and fibrosis even after prolonged POL, and is associated with constitutive activation of prohypertrophy and prosurvival signaling molecules such as phosphorylated GSK3beta, AKT, and ERK1/2 (25). Integrin-linked kinase (ILK) is a cytoplasmic scaffolding kinase that bridges the cytoplasmic domain of the beta1 integrin to the actin cytoskeleton via the parvins (26, 27). Genetic ablation of ILK in cardiac muscle results in spontaneous DCM and heart failure, and is associated with decreased beta1 integrin expression and reduced phosphorylation of FAK, AKT, ERK1/2, and p70S6K (26, 28-30). ILK is upregulated in cardiac hypertrophy and enhanced ILK signaling is associated with enhanced activation of Rac1, ERK1/2, p70S6K and p38, mildly decreased GSK3beta activity, and a compensated hypertrophic phenotype (26, 30). In a dog model of atrioventricular block, the acute cardiohypertrtophic response was associated with increases in beta1 and melusin expression and phosphorylation of AKT and GSK3beta (31). Overall cardiohypertrophic signaling from the beta1 integrin results in the phosphorylation of FAK, ERk1/2, AKT, mTOR, and GSK3beta, increased synthesis of contractile proteins and components of costameres and intercalated discs, as well as further expression of the alpha1, alpha5, alpha7 and beta 1 integrin subunits (9, 15, 18).

Several lines of evidence suggest that the predominant integrin mediating mechanosensory information to cardiomyocytes is the alpha7B (spliced with X1 or X2) beta1D integrin; [1] the alpha7B integrin is the exclusive partner for beta1D (4, 5), [2] the beta1D isoform comprises >95% of the beta integrin expressed on cardiomyocytes in vivo (4), [3] molecules that bind the beta1 integrin cytoplasmic domain communicate stretch signals and pro-hypertrophic responses (27), [4] cardiac specific reduction of beta1 integrin results in the development of DCM (13-16), and [5] alpha7Bbeta1D resides in the appropriate points of cellular attachment to "sense" the stretch of cardiomyocytes induced by an increased cardiac workload (2, 18, 32, 33). In addition to a mechanosensory role the alpha7beta1 integrin also contributes substantially to the structural integrity of skeletal and cardiac muscle. Humans and mice that do not express the alpha7 integrin manifest a mild muscular dystrophy with no apparent detriment in cardiac function (34-36). Alpha7 integrin and dystrophin double KO mice (mdx/alpha7 -/- dKO) exhibit a substantially more severe form of muscular dystrophy than mdx mice, including a mononuclear infiltration of the myocardium and cardiac myofibrillar disarray (37, 38). Transgenic overexpression of the alpha7X2B integrin in skeletal and cardiac tissues of severely dystrophic dystrophin and utrophin deficient (mdx/utro-/- dKO) mice ameliorated kyphosis and joint contractures, reduced cardiomyopathic lesions and ANF expression, reduced uptake of Evans blue dye into cardiomyocytes, and improved the life span of the mdx/utro-/- dKO mice from a median age of 12 weeks to 38 weeks (39-42). Thus, the alpha7Bbeta1D/ILK/melusin signaling axis is an important structural component of cardiomyocytes and mediator of cardiomyocyte adaptation to pressure overload, and a therapeutic approach based upon enhanced beta1 expression may attenuate the development of DCM in DMD patients.

DYSTROPHIN-DEFICIENT DILATED CARDIOMYOPATHY. Duchenne muscular dystrophy (DMD) affects 1 in 3500 male births and is caused by a deficiency of dystrophin, a large multifunctional cytoskeletal protein normally present within skeletal and cardiac muscle (43). The absence of dystrophin in patients with DMD results in a progressive deterioration of skeletal muscle as well as the development of DCM. Female carriers of DMD and Becker muscular dystrophy patients are at increased risk of DCM later in life, and a cardiac specific absence of dystrophin in patients with X-Linked DCM results in a rapid deterioration of heart function (44). Dystrophin links the contractile apparatus to the laminins of the extracellular matrix and protects the muscle membrane from the inherent shearing forces of muscle contraction. The N-terminal actin-binding domain of dystrophin binds the contractile apparatus, a central hinge and rod domain acts as a molecular shock absorber, and the C-terminal domain tethers dystrophin to beta-dystroglycan (45-47). The alpha and beta dystroglycan heterodimer is a laminin receptor, and in association with the sarcoglycans, comprise the dystrophin-associated proteins of the muscle membrane. Laminin-211 (LAM-211) is a predominant component of the cardiomyocyte basal lamina and binds both the glycosylated extracellular portion of alpha dystroglycan as well as the alpha7X1beta1 and alpha7X2beta1 integrins (19, 20, 48).

One major etiological basis of dystrophin-deficient DCM is a loss of cellular adhesion. The absence of dystrophin in cardiac muscle results in a secondary loss of alpha and beta dystroglycan and the loss of the primary linkage between the intracellular contractile apparatus and LAM-211 of the basal lamina (49, 50). As a consequence cardiac muscles of DMD patients are abnormally sensitive to damage, myofibers constitutively "sense" they are overworked, even under conditions of mild exercise or activity, and attempt to adapt through hypertrophic compensation (51-57). As DMD patients become older, muscle function begins to decline, patients lose the ability to walk, feed and breathe without assistance, and the quality of life becomes extremely poor (43). The most effective near-term therapies for DMD patients are likely to include the dystrophin mRNA rescue technologies (58, 59), agents that increase expression of molecules such as utrophin and alpha7 integrin, and intravenous protein therapies that recruit utrophin and alpha7 integrin to the sarcolemma (60, 61). Each of these therapeutic approaches restores the direct linkage between the contractile apparatus, muscle sarcolemma, and basal lamina through different mechanisms of action, and given FDA approval, simultaneous application of each technology to DMD patients should provide multiple layers of benefit. DMD patients usually succumb to respiratory collapse within their third decade of life, but improved therapies are allowing patients to live longer (62). Unfortunately this will impart greater demands on the heart and increased penetrance of the DCM in DMD patients is expected to increase (63). Treatment with beta-blockers and ACE inhibitors can delay the progression of dystrophin-deficient DCM and has a beneficial effect on long-term survival of DMD patients (64). These drugs do not improve the structural integrity of cardiomyocytes however, and current data suggests that the ability of the dystrophin rescue technologies to restore cardiac dystrophin is less efficient than that of skeletal muscle (58, 59, 65, 66). Thus, there is a critical need for therapeutics that can restore the structural integrity of both skeletal and cardiac muscles of DMD patients, including those patients that cannot respond to the dystrophin rescue technologies.

THE BASAL LAMINA OF NORMAL AND DYSTROPHIC MYOCARDIUM. Cardiomyocytes primarily synthesize collagen types IV and VI, laminins, and proteoglycans while interstitial fibroblasts primarily produce collagen types I and III, fibronectin, and metalloproteinases (8) The laminin isoform LAM-211 (alpha2beta1gamma1) is the predominant laminin isoform surrounding cardiomyocytes and is primarily a ligand for alpha-dystroglycan and the alpha7Bbeta1D integrin (19, 20, 61). LAM-511 appears around cardiomyocytes to a much lesser extent than LAM-211, and LAM-411 and -511 also appear around blood vessels (32, 67, 68). In animal models of induced hypertension, the myocardial levels of collagen types I and III, and fibronectin are increased, while the levels of laminin are reported to mildly increase or not increase at all (69-72), and the accumulation of collagens I and III and fibronectin are the greatest overall contributors to pathological fibrosis of the myocardium (8). Thus, following pressure overload the increased surface area of hypertrophic cardiomyocytes and increased expression of laminin-binding integrins are not matched by a commensurate increase in LAM-211 expression. Given that LAM-211 is not the optimal ligand for the alpha7beta1 integrin may further exasperate the pathological remodeling of the myocardium during DCM (8, 19, 20). Unfortunately for DMD patients, the reduction of alpha and beta dystroglycan in dystrophin-deficient cardiac muscle results in the additional loss of LAM-211 binding and further exasperates the development of DCM.

Among the four isoforms of the alpha7 integrin that are expressed in cardiac muscles, the alpha7X2B isoform predominates and is also the isoform when transgenically expressed rescues severely dystrophic mdx/utro-/- mice (2, 21, 39, 40). The X1 or X2 extracellular domain of the alpha7 integrin dictates the affinity for various laminin isoforms and in vitro analysis of the affinities of the laminin-binding integrins alpha3beta1, alpha6beta1, alpha7X1beta1, and alpha7X2beta1 towards various laminin isoforms demonstrated that LAM-211 binds to alpha7X1beta1 and alpha7X2beta1 with roughly equal affinity, but the alpha7X2Bbeta1 integrin isoform has the highest affinity for LAM-111, while the alpha7X1Bbeta1 integrin has the highest affinity for LAM-511 (19, 20, 73-75). Normal human and mouse myotubes grown in culture bound equally well to LAM-111 and LAM-211/221, while DMD and mdx myotubes adhered to LAM-111 to a far greater extent than to LAM-211/221, and the binding of DMD and mdx myotubes to LAM-111 was completely inhibited by anti-beta1 integrin antibody (beta1 was presumably paired with alpha7 in both cell types) (76). LAM-111 is not expressed in normal or dystrophic cardiac muscle, yet the preponderance of evidence shows that compared to LAM-211, LAM-111 is a preferred ligand for the alpha7X2Bbeta1 integrin (19, 20, 67, 77). Early work employing blot overlay experiments demonstrated that alpha dystroglycan bound LAM-111 more avidly than LAM-211 (47, 78), but more recent experiments with recombinant material suggests that the affinity of alpha-dystroglycan for LAM-111 is marginally lower than its affinity for LAM-211 (79, 80).

Dystrophin-deficient cardiomyocytes attempt to compensate for the absence of dystrophin through natural enhancement of utrophin and alpha7beta1 integrin expression as well as enhanced sarcolemma localization of each molecule following engagement to unbound LAM-211 (81-86). Unfortunately, the naturally increased recruitment and redistribution of utrophin and alpha7beta1 integrin in mdx mice or DMD patients is not sufficient to prevent the progression of disease. Indeed studies in transgenic mice suggest the levels of utrophin or alpha7beta1 integrin would need to be present at superphysiologic levels to rescue the dystrophic phenotype of DMD patients (39, 40, 87). Characterization of mdx/utro -/- dKO mouse expressing transgenic (rat) alpha7X2B integrin demonstrated that alpha7A was unchanged and alpha7B was increased by ~150% (utrophin is absent due to the knockout) (40). Endogenous mouse alpha7 integrin expression is equivalent in mdx and mdx/utro -/- dKO mice, and thus the amelioration of the dystrophic phenotype in transgenic alpha7X2B mdx/utro -/- dKO mice occurred solely from the 150% increase in transgenic rat alpha7X2B (40).

LAM-111: AN INTRAVENOUS PROTEIN THERAPY FOR DYSTROPHIN-DEFICIENT DCM. The ability of alpha7X2B integrin overexpression to ameliorate the skeletal and cardiac manifestations of disease in severely dystrophic mdx/utro-/- dKO mice, and the preference of the alpha7X2Bbeta1 integrin for LAM-111 versus LAM-211 suggests that an intravenous LAM-111 therapy would represent a novel therapeutic approach for treatment of dystrophin-deficient DCM (19, 20, 39, 40).

LONG-TERM IMPLICATIONS OF BETA1 INTEGRIN ENGAGEMENT. For those patients with dystrophin deficiency, a strategy that upregulates molecules that functionally compensate for the absence of dystrophin and focus hypertrophic signals towards cardiomyocytes rather than neighboring interstitial cells would represent a more selective approach to maintain a compensatory hypertrophic state (8). It is unlikely that this approach would prevent the release of hypertrophic signals from cardiomyocytes or interstitial cells, but over the lifetime of the patient, any mitigation of such paracrine and autocrine cross-talk may delay the onset or progression to a decompensatory state and heart failure. Laminins (LAM-211 or LAM-111) are not considered contributors to myocardial fibrosis, but we must also consider that deposition of exogenous LAM-111 around cardiomyocytes may negatively affect myocardial remodeling. However, given the beneficial effect of LAM-111 on the structural integrity of mdx skeletal muscle, such a side effect if it does indeed occur may be a tolerable risk. Given that therapeutic targeting of the alpha7beta1 integrin will occur over the lifetime of the DMD patient, we must consider the consequences of chronic activation of the beta1 integrin in cardiomyocytes. As demonstrated in numerous transgenic and knockout animal models of cardiac hypertrophy, the intensity and duration of a given signaling pathway, the amount of intersection among different signaling pathways, and whether various intersecting signaling pathways neutralize or synergize each other will certainly influence the path to compensatory hypertrophy, or conversely to heart failure (1). Therefore, only empirical testing will determine if chronic activation of the ILK/melusin/FAK/AKT/MAPK signaling axis following administration of LAM-111 is tolerable, whether enhanced expression of the alpha7beta1 integrin can functionally compensate for the absence of dystrophin, and whether a compensatory hypertrophic state of the DMD heart can be maintained.

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