Expression and function of four AAV-based constructs for dystrophin restoration in the mdx mouse model of Duchenne muscular dystrophy.

Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared the efficacies of four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e. reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement.

Visualizing Muscle Sialic Acid Expression in the GNED207VTgGne-/- Cmah-/- Model of GNE Myopathy: A Comparison of Dietary and Gene Therapy Approaches.

BACKGROUND: GNE myopathy (GNEM) is a rare, adult-onset, inclusion body myopathy that results from mutations in the GNE gene. GNE encodes UDP-GlcNAc epimerase/ManNAc-6 kinase, a protein with two enzymatic activities that comprise the committed step in biosynthesis of sialic acid (SA), an essential glycan that appears on the terminal positions of many extracellular oligosaccharide chains. These GNE mutations can cause a reduction of SA in many tissues, although pathology is restricted to skeletal muscles through a poorly understood mechanism. OBJECTIVE: Despite recent advances in the field, it remains unclear which therapeutic avenue is most promising for the restoration of SA level in skeletal muscle affected by GNEM. Our objective was to assess dietary and gene therapy strategies for GNEM in Cmah-deficient GNED207VTgGne-/- mice, a model that allows for the visualization of orally delivered N-glycolylneuraminic acid (Neu5Gc), one of the two predominant SA forms in muscle. METHODS: Methods included in situ physiology studies of the tibialis anterior muscle, studies of ambulation and limb grip strength, and muscle staining using MAA, SNA, and anti-Neu5Gc antibody, along with qPCR, qRT-PCR, western blot, and HPLC studies to assess virally introduced DNA, GNE gene expression, GNE protein expression, and SA expression. RESULTS: We found that a diet enriched in Neu5Gc-containing glycoproteins had no impact on Neu5Gc immunostaining in muscles of GNEM model mice. Delivery of a single high dose oral Neu5Gc therapy, however, did increase Neu5Gc immunostaining, though to levels below those found in wild type mice. Delivery of a single dose of GNE gene therapy using a recombinant Adeno Associated Virus (rAAV) vector with a liver-specific or a muscle-specific promoter both caused increased muscle Neu5Gc immunostaining that exceeded that seen with single dose monosaccharide therapy. CONCLUSIONS: Our findings indicate that dietary loading of Neu5Gc-containing glycoproteins is not effective in increasing muscle Neu5Gc expression, while single dose oral Neu5Gc monosaccharide or GNE gene therapy are. Neu5Gc immunostaining, however, showed greater changes than did lectin staining or HPLC analysis. Taken together, these results suggest that Neu5Gc immunostaining may be more sensitive technique to follow SA expression than other more commonly used methods and that liver expression of GNE may contribute overall muscle SA content.

Dose-Escalation Study of Systemically Delivered rAAVrh74.MHCK7.micro-dystrophin in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by mutations in the DMD gene. More than 2,000 mutations of the DMD gene are responsible for progressive loss of muscle strength, loss of ambulation, and generally respiratory and cardiac failure by age 30. Recently, gene transfer therapy has received widespread interest as a disease-modifying treatment for all patients with DMD. We designed an adeno-associated virus vector (rAAVrh74) containing a codon-optimized human micro-dystrophin transgene driven by a skeletal and cardiac muscle-specific promoter, MHCK7. To test the efficacy of rAAVrh74.MHCK7.micro-dystrophin, we evaluated systemic injections in mdx (dystrophin-null) mice at low (2 × 1012 vector genome [vg] total dose, 8 × 1013 vg/kg), intermediate (6 × 1012 vg total dose, 2 × 1014 vg/kg), and high doses (1.2 × 1013 vg total dose, 6 × 1014 vg/kg). Three months posttreatment, specific force increased in the diaphragm (DIA) and tibialis anterior muscle, with intermediate and high doses eliciting force outputs at wild-type (WT) levels. Histological improvement included reductions in fibrosis and normalization of myofiber size, specifically in the DIA, where results for low and intermediate doses were not significantly different from the WT. Significant reduction in central nucleation was also observed, although complete normalization to WT was not seen. No vector-associated toxicity was reported either by clinical or organ-specific laboratory assessments or following formal histopathology. The findings in this preclinical study provided proof of principle for safety and efficacy of systemic delivery of rAAVrh74.MHCK7.micro-dystrophin at high vector titers, supporting initiation of a Phase I/II safety study in boys with DMD.

Preclinical Systemic Delivery of Adeno-Associated α-Sarcoglycan Gene Transfer for Limb-Girdle Muscular Dystrophy.

Limb-girdle muscular dystrophy type 2D/R3 (LGMD2D/R3) is a progressive muscular dystrophy that manifests with muscle weakness, respiratory abnormalities, and in rare cases cardiomyopathy. LGMD2D/R3 is caused by mutations in the SGCA gene resulting in loss of protein and concomitant loss of some or all components of the dystrophin-associated glycoprotein complex. The sgca-null (sgca-/-) mouse recapitulates the clinical phenotype of patients with LGMD2D/R3, including dystrophic features such as muscle necrosis and fibrosis, elevated serum creatine kinase (CK), and reduction in the generation of absolute muscle force and locomotor activity. Thus, sgca-/- mice provide a relevant model to test the safety and efficacy of gene transfer. We designed a self-complementary AAVrh74 vector containing a codon-optimized full-length human SGCA (hSGCA) transgene driven by a muscle-specific promoter, shortened muscle creatine kinase (tMCK). In this report, we test the efficacy and safety of scAAVrh74.tMCK.hSGCA in sgca-/- mice using a dose-escalation design to evaluate a single systemic injection of 1.0 × 1012, 3.0 × 1012, and 6.0 × 1012 vg total dose compared with vehicle-treatment and wild-type mice. In sgca-/- mice, treatment with scAAVrh74.tMCK.hSGCA resulted in robust expression of α-sarcoglycan protein at the sarcolemma membrane in skeletal muscle at all doses tested. In addition, scAAVrh74.tMCK.hSGCA was effective in improving the histopathology of limb and diaphragm muscle of sgca-/- mice, as indicated by reductions in fibrosis, central nucleation, and normalization of myofiber size. These molecular changes were concomitant with significant increases in specific force generation in the diaphragm and tibialis anterior muscle, protection against eccentric force loss, and reduction in serum CK. Locomotor activity was improved at all doses of vector-treated compared with vehicle-treated sgca-/- mice. Lastly, vector toxicity was not detected in a serum chemistry panel and by gross necropsy. Collectively, these findings provide support for a systemic delivery of scAAVrh74.tMCK.hSGCA in a clinical setting for the treatment of LGMD2D/R3.

AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model - the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.

Systemic Delivery of Dysferlin Overlap Vectors Provides Long-Term Gene Expression and Functional Improvement for Dysferlinopathy.

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin.

Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency resulting in progressive muscle weakness and fibrotic scarring. Muscle fibrosis impairs blood flow, hampering muscle repair and regeneration. Irrespective of the success of gene restoration, functional improvement is limited without reducing fibrosis. The levels of miR-29c, a known regulator of collagen, are reduced in DMD. Our goal is to develop translational, antifibrotic therapy by overexpressing miR-29c. We injected the gastrocnemius muscle with either self-complementary AAV.CMV.miR-29c or single-stranded AAV.MCK.micro-dystrophin alone or in combination in the mdx/utrn+/- mouse, a DMD mouse model. Treatment of 3-month-old mdx/utrn+/- mice with AAV.miR-29c showed a reduction in collagen and increased absolute and specific force compared with untreated animals, but neither parameter reached WT levels. Combinatorial gene delivery in 3-month-old mdx/utrn+/- mice further decreased fibrosis, and showed a reduction of transcript levels for Col1A, Col3A, fibronectin, and Tgfb1. In addition, absolute and specific force was normalized and equivalent to WT. However, protection against eccentric contraction fell short of WT levels at this time point. When this same mouse model was treated with miR-29c/micro-dystrophin combinatorial therapy at 1 month of age, there was complete normalization of specific and absolute force and protection against eccentric contraction-induced injury was comparable to WT. These findings highlight the potential for miR-29c as an important addition to the armamentarium for translational gene therapy, especially when used in combination with micro-dystrophin in DMD.

Systemic AAV-Mediated ß-Sarcoglycan Delivery Targeting Cardiac and Skeletal Muscle Ameliorates Histological and Functional Deficits in LGMD2E Mice.

Limb-girdle muscular dystrophy type 2E (LGMD2E), resulting from mutations in ß-sarcoglycan (SGCB), is a progressive dystrophy with deteriorating muscle function, respiratory failure, and cardiomyopathy in 50% or more of LGMD2E patients. SGCB knockout mice share many of the phenotypic deficiencies of LGMD2E patients. To investigate systemic SGCB gene transfer to treat skeletal and cardiac muscle deficits, we designed a self-complementary AAVrh74 vector containing a codon-optimized human SGCB transgene driven by a muscle-specific promoter. We delivered scAAV.MHCK7.hSGCB through the tail vein of SGCB-/- mice to provide a rationale for a clinical trial that would lead to clinically meaningful results. This led to 98.1% transgene expression across all muscles that was accompanied by improvements in histopathology. Serum creatine kinase (CK) levels were reduced following treatment by 85.5%. Diaphragm force production increased by 94.4%, kyphoscoliosis of the spine was significantly reduced by 48.1%, overall ambulation increased by 57%, and vertical rearing increased dramatically by 132% following treatment. Importantly, no adverse effects were seen in muscle of wild-type mice injected systemically with scAAV.hSGCB. In this well-defined model of LGMD2E, we have demonstrated the efficacy and safety of systemic scAAV.hSGCB delivery, and these findings have established a path for clinically beneficial AAV-mediated gene therapy for LGMD2E.

Illuminating the Signals Job Seekers Receive from an Employer's Community Involvement and Environmental Sustainability Practices: Insights into Why Most Job Seekers Are Attracted, Others Are Indifferent, and a Few Are Repelled.

Evidence shows that job seekers tend to be attracted to employers known for their corporate social responsibility (CSR), but relatively little is known about the underlying psychological processes. Moreover, the literature is silent about whether and why some job seekers are unaffected, or even repelled by, an employer's CSR. We conducted a substantive replication of recent empirical support for three signal-based mechanisms by adapting the experimental manipulation used in a prior study while employing an alternative approach to analyzing a distinctly different type of data. We also extended prior work by examining other possible explanatory mechanisms and exploring potentially negative reactions to CSR. Using signaling theory as an overarching framework, we assessed research questions and tested hypotheses grounded in theories of employee recruitment and the psychology of CSR, specifying how an employer's CSR practices send signals from which job seekers draw inferences about unknown working conditions, thereby affecting their attraction to the employer. Study participants (N = 108) reviewed the webpages of two hiring companies and responded to open-ended questions about each employer. We content-analyzed written responses pertaining to one employer's webpages in which we embedded an experimental manipulation of information about the employer's community involvement or its environmentally sustainable practices. The results supported hypotheses that corroborate prior evidence for the "perceived value fit" and "expected employee treatment" mechanisms, and provided some, but relatively limited, support for the "anticipated pride" mechanism. Assessment of research questions highlighted previously undiscovered signal-based mechanisms that might help explain job seekers' attraction to CSR (e.g., inferences about the employer's positive work environment and financial standing, and the nature of its employees). Results also showed that a few people were less attracted because of the employer's CSR practices. Analyses among those individuals, combined with one-third of the sample who reported their attraction was unaffected by the employer's CSR, provided insights about when and why CSR fails to enhance attraction, such as when job seekers focus on other priorities, or are deeply skeptical and cynical about the employer's CSR. We discuss the implications for advancing a signal-based theory of CSR and employee recruitment, and recruitment practice.

Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy.

Limb-girdle muscular dystrophies are a genetically diverse group of diseases characterized by chronic muscle wasting and weakness. Recessive mutations in ANO5 (TMEM16E) have been directly linked to several clinical phenotypes including limb-girdle muscular dystrophy type 2L and Miyoshi myopathy type 3, although the pathogenic mechanism has remained elusive. ANO5 is a member of the Anoctamin/TMEM16 superfamily that encodes both ion channels and regulators of membrane phospholipid scrambling. The phenotypic overlap of ANO5 myopathies with dysferlin-associated muscular dystrophies has inspired the hypothesis that ANO5, like dysferlin, may be involved in the repair of muscle membranes following injury. Here we show that Ano5-deficient mice have reduced capacity to repair the sarcolemma following laser-induced damage, exhibit delayed regeneration after cardiotoxin injury and suffer from defective myoblast fusion necessary for the proper repair and regeneration of multinucleated myotubes. Together, these data suggest that ANO5 plays an important role in sarcolemmal membrane dynamics. Genbank Mouse Genome Informatics accession no. 3576659.

Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD.

AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models.

OBJECTIVE: Dysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment. METHODS: Traditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety. RESULTS: Treated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns. INTERPRETATION: Treated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients.

Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice.

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.

Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin α2 surrogates.

Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.

Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model.

Pharmacologic strategies have provided modest improvement in the devastating muscle-wasting disease, Duchenne muscular dystrophy (DMD). Pre-clinical gene therapy studies have shown promise in the mdx mouse model; however, studies conducted after disease onset fall short of fully correcting muscle strength or protecting against contraction-induced injury. Here we examine the treatment effect on muscle physiology in aged dystrophic mice with significant disease pathology by combining two promising therapies: micro-dystrophin gene replacement and muscle enhancement with follistatin, a potent myostatin inhibitor. Individual treatments with micro-dystrophin and follistatin demonstrated marked improvement in mdx mice but were insufficient to fully restore muscle strength and response to injury to wild-type levels. Strikingly, when combined, micro-dystrophin/follistatin treatment restored force generation and conferred resistance to contraction-induced injury in aged mdx mice. Pre-clinical studies with miniature dystrophins have failed to demonstrate full correction of the physiological defects seen in mdx mice. Importantly, the addition of a muscle enhancement strategy with delivery of follistatin in combination with micro-dystrophin gene therapy completely restored resistance to eccentric contraction-induced injury and improved force. Eccentric contraction-induced injury is a pre-clinical parameter relevant to the exercise induced injury that occurs in DMD patients, and herein, we demonstrate compelling evidence for the therapeutic potential of micro-dystrophin/follistatin combinatorial therapy.

Identification of M. tuberculosis thioredoxin reductase inhibitors based on high-throughput docking using constraints.

A virtual screening campaign is presented that led to small molecule inhibitors of thioredoxin reductase of Mycobacterium tuberculosis (MtTrxR) that target the protein-protein interaction site for the substrate thioredoxin (Trx). MtTrxR is a promising drug target because it dominates the Trx-dependent hydroperoxide metabolism and the reduction of ribonucleotides, thus facilitating survival and proliferation of M. tuberculosis. Moreover, MtTrxR sufficiently differs from its human homologs to suggest the possibility of selective inhibition if the MtTrxR-Trx interaction site is targeted. To this end, high-throughput docking of 6.5 million virtual compounds to the thioredoxin binding site of MtTrxR combined with constraints as filtering steps was applied. A total of 170 high-scoring compounds yielded 18 compounds that inhibited MtTrxR with IC50 values up to the low micromolar range, thus revealing that the protein-protein interaction site of MtTrxR is indeed druggable. Most importantly, selectivity toward MtTrxR in comparison to human TrxR (HsTrxR) is also demonstrated.

AAV-mediated overexpression of human α7 integrin leads to histological and functional improvement in dystrophic mice.

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene, with loss of its gene product, dystrophin. Dystrophin helps link integral membrane proteins to the actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We investigated an alternative therapeutic approach to dystrophin replacement by overexpressing human α7 integrin (ITGA7) using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal and cardiac muscle that links the extracellular matrix (ECM) to the actin skeleton. It is modestly upregulated in DMD muscle and has been proposed to be an important modifier of dystrophic symptoms. We delivered rAAV8.MCK.ITGA7 to the lower limb of mdx mice through isolated limb perfusion (ILP) of the femoral artery. We demonstrated ~50% of fibers in the tibialis anterior (TA) and extensor digitorum longus (EDL) overexpressing α7 integrin at the sarcolemma following AAV gene transfer. The increase in ITGA7 in skeletal muscle significantly protected against loss of force following eccentric contraction-induced injury compared with untreated (contralateral) muscles while specific force following tetanic contraction was unchanged. Reversal of additional dystrophic features included reduced Evans blue dye (EBD) uptake and increased muscle fiber diameter. Taken together, this data shows that rAAV8.MCK.ITGA7 gene transfer stabilizes the sarcolemma potentially preserving mdx muscle from further damage. This therapeutic approach demonstrates promise as a viable treatment for DMD with further implications for other forms of muscular dystrophy.

Characterization of bone resorption in novel in vitro and in vivo models of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vitro co-culture techniques. In vivo bioluminescent imaging, Faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor κB ligand (RANKL) expression and reduced osteoprotegerin (OPG) expression in conditioned medium from bone-invasive SCCF2 cells compared to minimally bone invasive SCCF3 cells, which was partially reversed with a neutralizing anti-PTHrP antibody. Human and feline OSCC cells cultured in bone-conditioned medium had increased PTHrP secretion and proliferation. Feline OSCC-induced bone resorption was associated with tumor cell secretion of PTHrP and with increased RANKL:OPG expression ratio in mouse preosteoblasts. Bone-CM increased OSCC proliferation and secretion of PTHrP. The preclinical models of feline OSCC recapitulated the bone-invasive phenotype characteristic of spontaneous OSCC and will be useful to future preclinical and mechanistic studies of bone invasive behavior.