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Partially decellularized tracheal scaffolds have emerged as a potential solution for long-segment tracheal defects. These grafts have exhibited regenerative capacity and the preservation of native mechanical properties resulting from the elimination of all highly immunogenic cell types while sparing weakly immunogenic cartilage. With partial decellularization, new considerations must be made about the viability of preserved chondrocytes. In this study, we propose a multimodal approach for quantifying chondrocyte viability for airway tissue engineering.Tracheal segments (5 mm) were harvested from C57BL/6 mice, and immediately stored in phosphate-buffered saline at -20°C (PBS-20) or biobanked via cryopreservation. Stored and control (fresh) tracheal grafts were implanted as syngeneic tracheal grafts (STG) for 3 months. STG was scanned with micro-computed tomography (μCT) in vivo. STG subjected to different conditions (fresh, PBS-20, or biobanked) were characterized with live/dead assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and von Kossa staining.Live/dead assay detected higher chondrocyte viability in biobanked conditions compared to PBS-20. TUNEL staining indicated that storage conditions did not alter the proportion of apoptotic cells. Biobanking exhibited a lower calcification area than PBS-20 in 3-month post-implanted grafts. Higher radiographic density (Hounsfield units) measured by μCT correlated with more calcification within the tracheal cartilage.We propose a strategy to assess chondrocyte viability that integrates with vivo imaging and histologic techniques, leveraging their respective strengths and weaknesses. These techniques will support the rational design of partially decellularized tracheal scaffolds.N/A Laryngoscope, 2022.

Synthetic scaffolds for the repair of long-segment tracheal defects are hindered by insufficient biocompatibility and poor graft epithelialization. In this study, we determined if extracellular matrix (ECM) coatings improved the biocompatibility and epithelialization of synthetic tracheal grafts (syn-TG). Porcine and human ECM substrates (pECM and hECM) were created through the decellularization and lyophilization of lung tissue. Four concentrations of pECM and hECM coatings on syn-TG were characterized for their effects on scaffold morphologies and on in vitro cell viability and growth. Uncoated and ECM-coated syn-TG were subsequently evaluated in vivo through the orthotopic implantation of segmental grafts or patches. These studies demonstrated that ECM coatings were not cytotoxic and, enhanced the in vitro cell viability and growth on syn-TG in a dose-dependent manner. Mass spectrometry demonstrated that fibrillin, collagen, laminin, and nephronectin were the predominant ECM components transferred onto scaffolds. The in vivo results exhibited similar robust epithelialization of uncoated and coated syn-TG patches, however, the epithelialization remained poor with either uncoated or coated scaffolds in the segmental replacement models. Overall, these findings demonstrated that ECM coatings improve the seeded cell biocompatibility of synthetic scaffolds in vitro, however, they do not improve graft epithelialization in vivo.

Objectives/hypothesis Tissue-engineered tracheal grafts (TETGs) offer a potential solution for repair of long-segment airway defects. However, preclinical and clinical TETGs have been associated with chronic inflammation and macrophage infiltration. Macrophages express great phenotypic heterogeneity (generally characterized as classically activated [M1] vs. alternatively activated [M2]) and can influence tracheal repair and regeneration. We quantified and characterized infiltrating host macrophages using mouse microsurgical tracheal replacement models. Study design Translational research, animal model. Methods We assessed macrophage infiltration and phenotype in animals implanted with syngeneic tracheal grafts, synthetic TETGs, or partially decellularized tracheal scaffolds (DTSs). Results Macrophage infiltration was observed following tracheal replacement with syngeneic trachea. Both M1 and M2 macrophages were present in native trachea and increased during early tracheal repair (P = .014), with an M1/M2 ratio of 0.48 ± 0.15. In contrast, orthotopic implantation of synthetic TETGs resulted in a shift to M1 predominant macrophage phenotype with an increased M1/M2 ratio of 1.35 ± 0.41 by 6 weeks following implant (P = .035). Modulation of the synthetic scaffold with the addition of polyglycolic acid (PGA) resulted in a reduction of M1/M2 ratio due to an increase in M2 macrophages (P = .006). Using systemic macrophage depletion, the M1/M2 ratio reverted to native values in synthetic TETG recipients and was associated with an increase in graft epithelialization. Macrophage ratios seen in DTSs were similar to native values. Conclusions M1 and M2 macrophages are present during tracheal repair. Poor epithelialization with synthetic TETG is associated with an elevation of the M1/M2 ratio. Macrophage phenotype can be altered with scaffold composition and host-directed systemic therapies. DTSs exhibit M1/M2 ratios similar to those seen in native trachea and syngeneic tracheal replacement. Level of evidence NA Laryngoscope, 2021.

Decellularized tracheal scaffolds offer a potential solution for the repair of long-segment tracheal defects. However, complete decellularization of trachea is complicated by tracheal collapse. We created a partially decellularized tracheal scaffold (DTS) and characterized regeneration in a mouse model of tracheal transplantation. All cell populations except chondrocytes were eliminated from DTS. DTS maintained graft integrity as well as its predominant extracellular matrix (ECM) proteins. We then assessed the performance of DTS in vivo. Grafts formed a functional epithelium by study endpoint (28 days). While initial chondrocyte viability was low, this was found to improve in vivo. We then used atomic force microscopy to quantify micromechanical properties of DTS, demonstrating that orthotopic implantation and graft regeneration lead to the restoration of native tracheal rigidity. We conclude that DTS preserves the cartilage ECM, supports neo-epithelialization, endothelialization and chondrocyte viability, and can serve as a potential solution for long-segment tracheal defects.

OBJECTIVES/HYPOTHESIS The ideal trachea replacement would be a living graft that is genetically identical to the host, avoiding the need for immunosuppression. We have developed a mouse model of syngeneic tracheal transplant that results in long-term survival without graft stenosis or delayed healing. To understand how host cells contribute to tracheal transplant integration, we quantified the populations of host cells in the graft and native trachea following implant. STUDY DESIGN Tracheal transplant, tracheal replacement, regenerative medicine, animal model. METHODS Tracheal grafts were obtained from female C57BL/6 mice and orthotopically transplanted into syngeneic male recipients. Cohorts were euthanized on day 14, day 45, and day 90 post-transplantation. Host and graft tracheas were explanted and analyzed by histology. Male host cells were quantified using fluorescence in situ hybridization, and macrophages were quantified with immunofluorescence. RESULTS Evidence of host-derived cells was found in the midgraft at the earliest time point (14 days). Host-derived cells transiently increased in the graft on day 45 and were predominantly found in the submucosa. By day 90, the population of host-derived cells population declined to a similar level on day 14. Macrophage infiltration of host and graft tissue was observed at all time points and was greatest on day 90. CONCLUSIONS Tracheal graft integration occurs by way of subacute transient host-cell infiltration and is primarily inflammatory in nature. Host-cell contribution to the graft epithelium is limited. These data indicate that creation of living, nonimmunogenic tracheal graft could serve as a viable solution for long-segment tracheal defects. LEVEL OF EVIDENCE 3 Laryngoscope, 131:E340-E345, 2021.

Background Previous studies in patients with limb-girdle muscular dystrophy type 2A (LGMD2A) have suggested that calpain-3 (CAPN3) mutations result in aberrant regeneration in muscle. Methods To gain insight into pathogenesis of aberrant muscle regeneration in LGMD2A, we used a paradigm of cardiotoxin (CTX)-induced cycles of muscle necrosis and regeneration in the CAPN3-KO mice to simulate the early features of the dystrophic process in LGMD2A. The temporal evolution of the regeneration process was followed by assessing the oxidative state, size, and the number of metabolic fiber types at 4 and 12 weeks after last CTX injection. Muscles isolated at these time points were further investigated for the key regulators of the pathways involved in various cellular processes such as protein synthesis, cellular energy status, metabolism, and cell stress to include Akt/mTORC1 signaling, mitochondrial biogenesis, and AMPK signaling. TGF-β and microRNA (miR-1, miR-206, miR-133a) regulation were also assessed. Additional studies included in vitro assays for quantifying fusion index of myoblasts from CAPN3-KO mice and development of an in vivo gene therapy paradigm for restoration of impaired regeneration using the adeno-associated virus vector carrying CAPN3 gene in the muscle. Results At 4 and 12 weeks after last CTX injection, we found impaired regeneration in CAPN3-KO muscle characterized by excessive numbers of small lobulated fibers belonging to oxidative metabolic type (slow twitch) and increased connective tissue. TGF-β transcription levels in the regenerating CAPN3-KO muscles were significantly increased along with microRNA dysregulation compared to wild type (WT), and the attenuated radial growth of muscle fibers was accompanied by perturbed Akt/mTORC1 signaling, uncoupled from protein synthesis, through activation of AMPK pathway, thought to be triggered by energy shortage in the CAPN3-KO muscle. This was associated with failure to increase mitochondria content, PGC-1α, and ATP5D transcripts in the regenerating CAPN3-KO muscles compared to WT. In vitro studies showed defective myotube fusion in CAPN3-KO myoblast cultures. Replacement of CAPN3 by gene therapy in vivo increased the fiber size and decreased the number of small oxidative fibers. Conclusion Our findings provide insights into understanding of the impaired radial growth phase of regeneration in calpainopathy. Electronic supplementary material The online version of this article (10.1186/s13395-017-0146-6) contains supplementary material, which is available to authorized users.

The ideal construct for tracheal replacement remains elusive in the management of long segment airway defects. Tissue engineered tracheal grafts (TETG) have been limited by the development of graft stenosis or collapse, infection, or lack of an epithelial lining. We applied a mouse model of orthotopic airway surgery to assess the impact of three critical barriers encountered in clinical applications: the scaffold, the extent of intervention, and the impact of cell seeding and characterized their impact on graft performance. First, synthetic tracheal scaffolds electrospun from polyethylene terephthalate / polyurethane (PET/PU) were orthotopically implanted in anterior tracheal defects of C57BL/6 mice. Scaffolds demonstrated complete coverage with ciliated respiratory epithelium by 2 weeks. Epithelial migration was accompanied by macrophage infiltration which persisted at long term (>6 weeks) time points. We then assessed the impact of segmental tracheal implantation using syngeneic trachea as a surrogate for the ideal tracheal replacement. Graft recovery involved local upregulation of epithelial progenitor populations and there was no evidence of graft stenosis or necrosis. Implantation of electrospun synthetic tracheal scaffold for segmental replacement resulted in respiratory distress and required euthanasia at an early time point. There was limited epithelial coverage of the scaffold with and without seeded bone marrow-derived mononuclear cells (BM-MNCs). We conclude that synthetic scaffolds support re-epithelialization in orthotopic patch implantation, syngeneic graft integration occurs with focal repair mechanisms, however epithelialization in segmental synthetic scaffolds is limited and is not influenced by cell seeding. STATEMENT OF SIGNIFICANCE: The life-threatening nature of long-segment tracheal defects has led to clinical use of tissue engineered tracheal grafts in the last decade for cases of compassionate use. However, the ideal tracheal reconstruction using tissue-engineered tracheal grafts (TETG) has not been clarified. We addressed the core challenges in tissue engineered tracheal replacement (re-epithelialization and graft patency) by defining the role of cell seeding with autologous bone marrow-derived mononuclear cells, the mechanism of respiratory epithelialization and proliferation, and the role of the inflammatory immune response in regeneration. This research will facilitate comprehensive understanding of cellular regeneration and neotissue formation on TETG, which will permit targeted therapies for accelerating re-epithelialization and attenuating stenosis in tissue engineered airway replacement.

Introduction Classic Charcot‐Marie‐Tooth (CMT) neuropathies including those with Schwann cell genetic defects exhibit a length‐dependent process affecting the distal axon. Energy deprivation in the distal axon has been the proposed mechanism accounting for length‐dependent distal axonal degeneration. We hypothesized that pyruvate, an intermediate glycolytic product, could restore nerve function, supplying lost energy to the distal axon. Methods To test this possibility, we supplied pyruvate to the drinking water of the Trembler‐J (TrJ) mouse and assessed efficacy based on histology, electrophysiology, and functional outcomes. Pyruvate outcomes were compared with untreated TrJ controls alone or adeno‐associated virus mediated NT‐3 gene therapy (AAV1.NT‐3)/pyruvate combinatorial approach. Results Pyruvate supplementation resulted increased myelinated fiber (MF) densities and myelin thickness in sciatic nerves. Combining pyruvate with proven efficacy from AAV1.tMCK.NT‐3 gene therapy provided additional benefits showing improved compound muscle action potential amplitudes and nerve conduction velocities compared to pyruvate alone cohort. The end point motor performance of both the pyruvate and the combinatorial therapy cohorts was better than untreated TrJcontrols. In a unilateral sciatic nerve crush paradigm, pyruvate supplementation improved myelin‐based outcomes in both regenerating and the contralateral uncrushed nerves. Conclusions This proof of principle study demonstrates that exogenous pyruvate alone or as adjunct therapy in TrJ may have clinical implications and is a candidate therapy for CMT neuropathies without known treatment.

Neurotrophin 3 (NT-3) has well-recognized effects on peripheral nerve and Schwann cells, promoting axonal regeneration and associated myelination. In this study, we assessed the effects of AAV.NT-3 gene therapy on the oxidative state of the neurogenic muscle from the TremblerJ (Tr J ) mice at 16 weeks post-gene injection and found that the muscle fiber size increase was associated with a change in the oxidative state of muscle fibers towards normalization of the fiber type ratio seen in the wild type. NT-3-induced fiber size increase was most prominent for the fast twitch glycolytic fiber population. These changes in the Tr J muscle were accompanied by increased phosphorylation levels of 4E-BP1 and S6 proteins as evidence of mTORC1 activation. In parallel, the expression levels of the mitochondrial biogenesis regulator PGC1α, and the markers of glycolysis (HK1 and PK1) increased in the TrJ muscle. In vitro studies showed that recombinant NT-3 can directly induce Akt/mTOR pathway activation in the TrkC expressing myotubes but not in myoblasts. In addition, myogenin expression levels were increased in myotubes while p75 NTR expression was downregulated compared to myoblasts, indicating that NT-3 induced myoblast differentiation is associated with mTORC1 activation. These studies for the first time have shown that NT-3 increases muscle fiber diameter in the neurogenic muscle through direct activation of mTOR pathway and that the fiber size increase is more prominent for fast twitch glycolytic fibers.

The spontaneous autoimmune peripheral polyneuropathy (SAPP) model in B7-2 knockout nonobese diabetic mice mimics a progressive and unremitting course of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In this study, bone marrow-derived dendritic cells (DCs) were transduced to express vasoactive intestinal polypeptide (VIP) using a lentiviral vector (LV-VIP). These transduced DCs (LV-VIP-DCs) were then injected intravenously (i.v.) into 16-week-old (before disease onset) and 21-week-old (after disease onset) SAPP mice in order to prevent or attenuate the disease. Outcome measures included behavioral tests, clinical and histological scoring, electrophysiology, real-time PCR, flow cytometry analyses, and enzyme-linked immunosorbent assay. LV-VIP-DCs were recruited to the inflamed sciatic nerve and reduced the expression of inflammatory cytokines. A single injection of LV-VIP-DC delayed the onset of disease, stabilized, and attenuated clinical signs correlating with ameliorated behavioral functions, reduced nerve demyelination, and improved nerve conduction. This proof-of-principle study is an important step potentially leading to a clinical translational study using DCs expressing VIP in cases of CIDP refractory to standard immunosuppressive therapy.

Duchenne muscular dystrophy is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus (AAV) will ultimately require a vascular approach to broadly transduce muscle cells. We tested the impact of preexisting AAV antibodies on microdystrophin expression following vascular delivery to nonhuman primates. Rhesus macaques were treated by isolated limb perfusion using a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of the three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus, and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Microdystrophin expression was visualized in AAV, rhesus serotype 74 sero-negative animals (mean: 48.0 ± 20.8%) that was attenuated in sero-positive animals (19.6 ± 18.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8 ± 18.0%), which is comparable with that of AAV sero-negative animals (53.7 ± 7.6%), whereas non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1 ± 6.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of preexisting immunity (natural infection) to AAV.

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.

Introduction Classic Charcot-Marie-Tooth (CMT) neuropathies including those with Schwann cell genetic defects exhibit a length-dependent process affecting the distal axon. Energy deprivation in the distal axon has been the proposed mechanism accounting for length-dependent distal axonal degeneration. We hypothesized that pyruvate, an intermediate glycolytic product, could restore nerve function, supplying lost energy to the distal axon. Methods To test this possibility, we supplied pyruvate to the drinking water of the Trembler-J (TrJ ) mouse and assessed efficacy based on histology, electrophysiology, and functional outcomes. Pyruvate outcomes were compared with untreated TrJ controls alone or adeno-associated virus mediated NT-3 gene therapy (AAV1.NT-3)/pyruvate combinatorial approach. Results Pyruvate supplementation resulted increased myelinated fiber (MF) densities and myelin thickness in sciatic nerves. Combining pyruvate with proven efficacy from AAV1.tMCK.NT-3 gene therapy provided additional benefits showing improved compound muscle action potential amplitudes and nerve conduction velocities compared to pyruvate alone cohort. The end point motor performance of both the pyruvate and the combinatorial therapy cohorts was better than untreated TrJ controls. In a unilateral sciatic nerve crush paradigm, pyruvate supplementation improved myelin-based outcomes in both regenerating and the contralateral uncrushed nerves. Conclusions This proof of principle study demonstrates that exogenous pyruvate alone or as adjunct therapy in TrJ may have clinical implications and is a candidate therapy for CMT neuropathies without known treatment.

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50-79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer,80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.

We have cloned the swine eNOS promoter and analyzed its function in newborn swine pulmonary artery endothelial cells (PAECs). Analysis of the 2.1 kb 5′ flanking region revealed that the swine eNOS promoter is, like its counterparts in human and other species, a TATA-less promoter. The transcription start site, determined by 5′ RLM-RACE, was located 62 bp upstream of the translation start codon. Promoter activity was demonstrated by transient transfection of 5′ deletion promoter/luciferase constructs into swine PAECs, and indicated that the proximal region from − 227 to − 82 was necessary for basal promoter activity. Positive cis-regulatory elements were present from − 227 to − 1290, while negative cis-regulatory elements may be present from − 1290 to − 1926 bp. Electrophoretic mobility shift assay (EMSA) of the proximal region demonstrated that multiprotein complexes were formed in the conserved proximal region of the swine eNOS promoter and a novel Spl site at − 68/ − 59 was involved in the formation of ...

The present study evaluated mechanical stretch-induced apoptosis in swine vascular smooth muscle cells (VSMC) of different phenotypes. We demonstrated that differentiated VSMC express a greater level of Bcl-2-associated death factor (BAD) and have a significant cell loss when exposed to mechanical stretch (10% elongation, 1 Hz) for 24 h. We further demonstrated that apoptosis was significantly increased only in differentiated VSMC exposed to mechanical stretch. To test the hypothesis that the intracellular level of BAD in VSMC determines its response to mechanical stretch-induced apoptosis, we examined whether BAD expression was upregulated by mechanical stretch-induced apoptosis and was associated with the increase in the apoptosis level of differentiated VSMC. When exposed to mechanical stretch, the expression of BAD in differentiated VSMC was elevated at 1 h and remained at higher levels during the application of stretch (24 h). In contrast, Bcl-2 expression was suppressed during the application of stretch. Moreover, the proapoptotic function of BAD was inhibited by overexpression of Bcl-2 through transient transfection of VSMC with pCEP4-Bcl-2 or incubation of VSMC with vascular epithelial growth factor. These results suggest that mechanical stretch-induced VSMC apoptosis is phenotype dependent. The higher levels of apoptosis of differentiated VSMC upon mechanical stretch were, at least in part, dependent on their intrinsic level of BAD.

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.

Duchenne Muscular Dystrophy (DMD) is a lethal X-linked disorder caused by truncating mutations in the DMD gene which result in an absence of the dystrophin protein. However, deletion mutations that preserve an open reading frame, lead to an internally truncated functional dystrophin which is associated with the milder Becker Muscular Dystrophy (BMD). This observation as led to exon-skipping therapies in DMD patients to restore the reading frame and generate a BMD phenotype. Our group has recently shown that mutations in the 5 ′ exons of the DMD gene that disrupt the open reading frame (ORF) induce expression of a functional N-truncated dystrophin via IRES-driven translation initiation within exon 6. We have previously shown that the exon 5 IRES can be activated by disruption of the ORF by AAV9. U7snRNA-mediated skipping of exon 2 by intramuscular (IM) or intravascular (IV) injection. We have now extended those studies to address the minimal efficacious dose (MED), a prerequisite for clinical development. To assess dose ranging with IM delivery, TA (tibialis anterior) injections were performed at 5 different doses, ranging from 1.1×10 10 to 1.1×10 12 total vector genomes (vg) in half-log steps ( N =6 TAs per dose). A gradient of exon 2 exclusion was seen by RT-PCR at all doses, but high levels of properly localized dystrophin expression were seen at 3.5×10 11 vg and above. Experiments to determine the correction of TA physiology at this dose are underway. Similarly, dose ranging experiments using IV tail vein injections with 5 doses ( N =3 per dose) ranging from 1×10 12 to 1×10 14 vg/kg are underway, and results of both protein expression and physiology studies will be presented. Our results show that U7snRNA-mediated exon skipping in Dup2 is both titratable and highly efficient. The establishment of a IV MED for this vector will allow planning of IND-enabling GLP toxicology studies.

We have cloned the swine eNOS promoter and analyzed its function in newborn swine pulmonary artery endothelial cells (PAECs). Analysis of the 2.1 kb 5' flanking region revealed that the swine eNOS promoter is, like its counterparts in human and other species, a TATA-less promoter. The transcription start site, determined by 5' RLM-RACE, was located 62 bp upstream of the translation start codon. Promoter activity was demonstrated by transient transfection of 5' deletion promoter/luciferase constructs into swine PAECs, and indicated that the proximal region from -227 to -82 was necessary for basal promoter activity. Positive cis-regulatory elements were present from -227 to -1290, while negative cis-regulatory elements may be present from -1290 to -1926 bp. Electrophoretic mobility shift assay (EMSA) of the proximal region demonstrated that multiprotein complexes were formed in the conserved proximal region of the swine eNOS promoter and a novel Spl site at -68/-59 was involved in the formation of these complexes.

DMD is a rare, degenerative, genetic disease that results in progressive muscle loss and premature death. Affecting 1 in 5000 male births, DMD is caused by the inability to produce the dystrophin protein. There are no approved drugs available to treat DMD, although glucocorticoids show a modest effect with long-term efficacy limited by side effects. Eteplirsen is an investigational therapy designed to enable functional dystrophin production in boys who are amenable to exon 51-skipping therapy (∼13%). Twelve boys aged 7–13 years with eligible genotypes were randomized 1:1:1 to 30 mg/kg, 50 mg/kg, or placebo. Upon completion of a 24-week double-blind, placebo-controlled study phase, all subjects were enrolled in an open-label extension and the placebo-treated subjects initiated eteplirsen treatment. The critical clinical endpoint was the change in 6-min walk test (6MWT) distance from baseline compared to the placebo/delayed-treatment cohort. Previously reported 48-week results showed a statistically significant increase in 6MWT distance for eteplirsen versus the placebo/delayed-treatment arm. At 62 weeks, a significant clinical benefit of 62 m was observed (p ⩽ 0.007) on the 6MWT for ambulatory-evaluable subjects in the combined eteplirsen-treated cohorts (n = 6) versus the placebo/delayed-treatment cohort (n = 4). The eteplirsen-treated cohorts showed less than a 5% decline in 6MWT distance from baseline. No clinically significant treatment-related adverse events were seen through 62 weeks. Through 62 weeks of treatment, eteplirsen demonstrated a significant clinical benefit on the 6MWT with no clinically significant treatment-related adverse events. Data on these outcomes will be reported through 2 years of treatment.

Becker muscular dystrophy (BMD), a dystrophinopathy with milder skeletal muscle manifestations compared to Duchenne muscular dystrophy, characteristically demonstrates weakness of knee extensors (KE), accompanied by muscle atrophy and fibrosis. The weakness predisposes to gait impairment and loss of ambulation. Myostatin, a member of the TGF-β superfamily of signal peptides, is expressed in skeletal muscle, acting through the activin type IIB (ACTRIIB) receptor. Follistatin, a myostatin antagonist, inhibits this pathway resulting in muscle hypertrophy, and improved muscle strength. FS315 and FS288 are two isoforms of follistatin generated by alternative splicing. FS315 has no cell-surface affinity providing a safety margin avoiding pituitary–gonadal axis complications. FS344 is the precursor cDNA to FS315. When FS344 is delivered to mdx mice in adeno-associated virus (AAV1. CMV. FS344), muscle mass and strength increase. Similar findings are seen in non-human primates without safety concerns. Six BMD patients, 18 years and older with proven in-frame DMD gene mutations were enrolled in a clinical trial. The first 3 subjects received 12 intramuscular (IM) injections of AAV1. CMV. FS344 at a total dose of 6e11 vg/kg in the quadriceps muscles bilaterally. A second cohort received the same number of IM injections at higher total dose (1.2e12 vg/kg). Studies extending beyond one year in Cohort 1 and for six months in Cohort 2 showed no adverse events. The distance walked (meters) on the 6-min walk test (6MWT) was the primary functional outcome measure. At one year the distance walked by two subjects in Cohort 1 increased by 125 m and 58 m; subject 3 improved modestly by 9 m. In Cohort 2, one patient reaching the 6-month time point increased by 108 meters. For the entire group of BMD patients only a single patient declined in distance. Preliminary analysis of muscle biopsies favor increased muscle regeneration contributing to improved function.

Eteplirsen, SRP-4045 and SRP-4053 are three PMOs developed as treatments for DMD. DMD is mostly caused by mutations in the dystrophin gene that lead to a reading frame shift and premature translation termination. PMOs promote exon skipping during splicing of dystrophin pre-mRNA, restoring the reading frame and translation of internally truncated, but functional dystrophin protein. Eteplirsen, SRP-4045, and SRP-4053 are intended to treat patients with mutations amenable to skipping exon 51, 45 and 53, respectively. IND enabling preclinical studies have been completed for eteplirsen, SRP-4045, and SRP-4053. In vitro metabolism data and PK profiles in animals were similar for all three PMOs. No adverse responses were detected in safety pharmacology studies in non-human primates (NHPs). Genotoxicity studies were negative. NHPs given 12 weekly IV doses up to 320 mg/kg showed no significant sequence-specific toxicities. A clinical study of eteplirsen with doses of 30 and 50 mg/kg/wk reported no clinically significant treatment-related adverse events through 120 weeks of treatment. A few cases of transient, mild urine protein elevation resolved without intervention or drug interruption and one femur fracture was determined to be treatment-unrelated. There were no hospitalizations, discontinuations, or clinically significant treatment-related changes for lab parameters, including liver enzymes, kidney function, coagulation, or platelet counts. Plasma Cmax and AUC increased proportionally with dose, and half-life was approximately 3 h with no plasma accumulation observed. The consistent preclinical profiles and lack of significant, sequence-specific toxicities for eteplirsen, SRP-4045, and SRP-4053 demonstrate PMOs to be a well-tolerated therapeutic class, with potential applications in a wide variety of disease indications.

DMD, a rare, degenerative, genetic disease that results in progressive muscle loss and premature death affects 1:5000 male births. It is caused by deletions in the dystrophin gene, which prevents production of the dystrophin protein. There are no approved treatments available, although corticosteroids have shown some benefit. Eteplirsen is an investigational drug designed to enable functional dystrophin production in boys who are amenable to skipping exon 51. Twelve boys aged 7–13years with eligible genotypes were randomized 1:1:1 to eteplirsen 30mg/kg/wk, 50mg/kg/wk, or placebo IV for 24-weeks. All patients transitioned into an ongoing open-label extension with 30 or 50mg/kg eteplirsen. Clinical efficacy endpoints included the 6-min walk test (6MWT) and pulmonary function testing. Safety assessments included adverse event recording, EKG, ECHO, hematology, blood chemistry and urinalysis. After 120weeks of treatment, a significant clinical benefit of 65m was observed ( p =0.006) on the 6MWT for ambulatory-evaluable patients in the combined eteplirsen-treated cohorts ( n =6) versus the placebo/delayed-treatment cohort ( n =4). The eteplirsen-treated cohorts showed a decline of less than 14m in walking ability from baseline, which was not statistically significant. After a substantial decline in the first 36weeks of the study, the placebo/delayed-treatment cohort demonstrated stabilization in walking ability at week 48, i.e., 12weeks after initiation of treatment with eteplirsen when meaningful levels of dystrophin were likely produced, with

Duchenne Muscular Dystrophy (DMD) is the most common, severe neuromuscular disorder caused by mutations in the DMD gene. 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 (ECM) to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and thus overexpression will not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically to the more severe mouse model of DMD, the mdx/utrn−/− mouse deficient for both dystrophin and utrophin at 2–4days of age. We demonstrated widespread expression of ITGA7 at the sarcolemma to multiple muscle groups following gene transfer eight weeks post injection. The increased expression of ITGA7 significantly extended longevity and reduced kyphosis and joint contractures, common to the mdx/utrn−/− mouse. The additional α 7 expression significantly protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles eight weeks post gene transfer. Taken together, these results show that this therapeutic approach continues to demonstrate promise as a viable treatment for DMD.