Airway disease decreases the therapeutic potential of epithelial stem cells.

BACKGORUND: Tissue-engineered tracheal grafts (TETG) can be recellularized by the host or pre-seeded with host-derived cells. However, the impact of airway disease on the recellularization process is unknown. METHODS: In this study, we determined if airway disease alters the regenerative potential of the human tracheobronchial epithelium (hTBE) obtained by brushing the tracheal mucosa during clinically-indicated bronchoscopy from 48 pediatric and six adult patients. RESULTS: Our findings revealed that basal cell recovery and frequency did not vary by age or region. At passage 1, all samples produced enough cells to cellularize a 3.5 by 0.5 cm2 graft scaffold at low cell density (~ 7000 cells/cm2), and 43.75% could cellularize a scaffold at high cell density (~ 100,000 cells/cm2). At passage 2, all samples produced the number of cells required for both recellularization models. Further evaluation revealed that six pediatric samples (11%) and three (50%) adult samples contained basal cells with a squamous basal phenotype. These cells did not form a polarized epithelium or produce differentiated secretory or ciliated cells. In the pediatric population, the squamous basal cell phenotype was associated with degree of prematurity (< 28 weeks, 64% vs. 13%, p = 0.02), significant pulmonary history (83% vs. 34%, p = 0.02), specifically with bronchopulmonary dysplasia (67% vs. 19%, p = 0.01), and patients who underwent previous tracheostomy (67% vs. 23%, p = 0.03). CONCLUSIONS: In summary, screening high-risk pediatric or adult population based on clinical risk factors and laboratory findings could define appropriate candidates for airway reconstruction with tracheal scaffolds. LEVEL OF EVIDENCE: Level III Cohort study.

Long-Term Chondrocyte Retention in Partially Decellularized Tracheal Grafts.

OBJECTIVE: Decellularized tracheal grafts possess the biological cues necessary for tissue regeneration. However, conventional decellularization approaches to target the removal of all cell populations including chondrocytes lead to a loss of mechanical support. We have created a partially decellularized tracheal graft (PDTG) that preserves donor chondrocytes and the mechanical properties of the trachea. In this study, we measured PDTG chondrocyte retention with a murine microsurgical model. STUDY DESIGN: Murine in vivo time-point study. SETTING: Research Institute affiliated with Tertiary Pediatric Hospital. METHODS: PDTG was created using a sodium dodecyl sulfate protocol. Partially decellularized and syngeneic grafts were orthotopically implanted into female C57BL/6J mice. Grafts were recovered at 1, 3, and 6 months postimplant. Pre- and postimplant grafts were processed and analyzed via quantitative immunofluorescence. Chondrocytes (SOX9+, DAPI+) present in the host and graft cartilage was evaluated using ImageJ. RESULTS: Partial decellularization resulted in the maintenance of gross tracheal architecture with the removal of epithelial and submucosal structures on histology. All grafts demonstrated SOX9+ chondrocytes throughout the study time points. Chondrocytes in PDTG were lower at 6 months compared to preimplant and syngeneic controls. CONCLUSION: PDTG retained donor graft chondrocytes at all time points. However, PDTG exhibits a reduction in chondrocytes at 6 months. The impact of these histologic changes on cartilage extracellular matrix regeneration and repair remains unclear.

Assessing the Biocompatibility and Regeneration of Electrospun-Nanofiber Composite Tracheal Grafts.

OBJECTIVE: Composite tracheal grafts (CTG) combining decellularized scaffolds with external biomaterial support have been shown to support host-derived neotissue formation. In this study, we examine the biocompatibility, graft epithelialization, vascularization, and patency of three prototype CTG using a mouse microsurgical model. STUDY DESIGN: Tracheal replacement, regenerative medicine, biocompatible airway splints, animal model. METHOD: CTG electrospun splints made by combining partially decellularized tracheal grafts (PDTG) with polyglycolic acid (PGA), poly(lactide-co-ε-caprolactone) (PLCL), or PLCL/PGA were orthotopically implanted in mice (N = 10/group). Tracheas were explanted two weeks post-implantation. Micro-Computed Tomography was conducted to assess for graft patency, and histological analysis was used to assess for epithelialization and neovascularization. RESULT: Most animals (greater than 80%) survived until the planned endpoint and did not exhibit respiratory symptoms. MicroCT confirmed the preservation of graft patency. Grossly, the PDTG component of CTG remained intact. Examining the electrospun component of CTG, PGA degraded significantly, while PLCL+PDTG and PLCL/PGA + PDTG maintained their structure. Microvasculature was observed across the surface of CTG and infiltrating the pores. There were no signs of excessive cellular infiltration or encapsulation. Graft microvasculature and epithelium appear similar in all groups, suggesting that CTG did not hinder endothelialization and epithelialization. CONCLUSION: We found that all electrospun nanofiber CTGs are biocompatible and did not affect graft patency, endothelialization and epithelialization. Future directions will explore methods to accelerate graft regeneration of CTG. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1155-1162, 2024.

Partial decellularization eliminates immunogenicity in tracheal allografts.

There is currently no suitable autologous tissue to bridge large tracheal defects. As a result, no standard of care exists for long-segment tracheal reconstruction. Tissue engineering has the potential to create a scaffold from allografts or xenografts that can support neotissue regeneration identical to the native trachea. Recent advances in tissue engineering have led to the idea of partial decellularization that allows for the creation of tracheal scaffolds that supports tracheal epithelial formation while preserving mechanical properties. However, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of partially decellularized tracheal grafts (PDTG) is a critical step toward clinical translation. Here, we determined that tracheal allograft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium and that partial decellularization creates grafts that are able to support an epithelium without histologic signs of rejection. Moreover, allograft implantation elicits CD8+ T-cell infiltration, a mediator of rejection, while PDTG did not. Hence, we establish that partial decellularization eliminates allograft immunogenicity while creating a scaffold for implantation that can support spatially appropriate airway regeneration.

Regeneration of tracheal neotissue in partially decellularized scaffolds.

Extensive tracheal injury or disease can be life-threatening but there is currently no standard of care. Regenerative medicine offers a potential solution to long-segment tracheal defects through the creation of scaffolds that support the generation of healthy neotissue. We developed decellularized tracheal grafts (PDTG) by removing the cells of the epithelium and lamina propria while preserving donor cartilage. We previously demonstrated that PDTG support regeneration of host-derived neotissue. Here, we use a combination of microsurgical, immunofluorescent, and transcriptomic approaches to compare PDTG neotissue with the native airway and surgical controls. We report that PDTG neotissue is composed of native tracheal cell types and that the neoepithelium and microvasculature persisted for at least 6 months. Vascular perfusion of PDTG was established within 2 weeks and the graft recruited multipotential airway stem cells that exhibit normal proliferation and differentiation. Hence, PDTG neotissue recapitulates the structure and function of the host trachea and has the potential to regenerate.

Optimization of Chondrocyte Viability in Partially Decellularized Tracheal Grafts.

OBJECTIVE: Advancements in tissue-engineered tracheal replacement (TETR) show promise for the use of partially decellularized tracheal grafts (PDTG) to address critical gaps in airway management and reconstruction. In this study, aiming to leverage the immunoprivileged nature of cartilage to preserve tracheal biomechanics, we optimize PDTG for retention of native chondrocytes. STUDY DESIGN: Comparison in vivo murine study. SETTING: Research Institute affiliated with Tertiary Pediatric Hospital. METHODS: PDTG were created per a shortened decellularization protocol using sodium dodecyl sulfate, then biobanked via cryopreservation technique. Decellularization efficiency was characterized by DNA assay and histology. Viability and apoptosis of chondrocytes in preimplanted PDTG and biobanked native trachea (control) was assessed with live/dead and apoptosis assays. PDTG (N = 5) and native trachea (N = 6) were orthotopically implanted in syngeneic recipients for 1-month. At the endpoint, microcomputed tomography (micro-CT) was employed to interrogate graft patency and radiodensity in vivo. Vascularization and epithelialization were qualitatively analyzed using histology images following explant. RESULTS: PDTG exhibited complete decellularization of all extra-cartilaginous cells and reduced DNA content compared to control. Chondrocyte viability and nonapoptotic cell populations were improved utilizing biobanking and shorter decellularization time. All grafts remained patent. Evaluation of graft radiodensity at 1 month revealed elevation of Hounsfield units in both PDTG and native compared to host, with PDTG showing higher radiodensity than native. PDTG supported complete epithelialization and functional reendothelialization 1-month postimplantation. CONCLUSION: Optimizing PDTG chondrocyte viability is a key component to successful tracheal replacement. Ongoing research seeks to evaluate the acute and chronic immunogenicity of PDTG.

A Multimodal Approach to Quantify Chondrocyte Viability for Airway Tissue Engineering.

OBJECTIVES/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. LEVEL OF EVIDENCE: N/A Laryngoscope, 133:512-520, 2023.

Biobanked tracheal basal cells retain the capacity to differentiate.

Objective: While airway epithelial biorepositories have established roles in the study of bronchial progenitor stem (basal) cells, the utility of a bank of tracheal basal cells from pediatric patients, who have or are suspected of having an airway disease, has not been established. In vitro study of these cells can enhance options for tracheal restoration, graft design, and disease modeling. Development of a functional epithelium in these settings is a key measure. The aim of this study was the creation a tracheal basal cell biorepository and assessment of recovered cells. Methods: Pediatric patients undergoing bronchoscopy were identified and endotracheal brush (N = 29) biopsies were collected. Cells were cultured using the modified conditional reprogramming culture (mCRC) method. Samples producing colonies by day 14 were passaged and cryopreserved. To explore differentiation potential, cells were thawed and differentiated using the air-liquid interface (ALI) method. Results: No adverse events were associated with biopsy collection. Of 29 brush biopsies, 16 (55%) were successfully cultured to passage 1/cryopreserved. Samples with higher initial cell yields were more likely to achieve this benchmark. Ten unique donors were then thawed for analysis of differentiation. The average age was 2.2 ± 2.2 years with five donors (50%) having laryngotracheal pathology. Nine donors (90%) demonstrated differentiation capacity at 21 days of culture, as indicated by detection of ciliated cells (ACT+) and mucous cells (MUC5B+). Conclusion: Pediatric tracheal basal cells can be successfully collected and cryopreserved. Recovered cells retain the ability to differentiate into epithelial cell types in vitro. Level of Evidence: Level 3.

Tissue-engineered composite tracheal grafts create mechanically stable and biocompatible airway replacements.

We tested composite tracheal grafts (CTG) composed of a partially decellularized tracheal graft (PDTG) combined with a 3-dimensional (3D)-printed airway splint for use in long-segment airway reconstruction. CTG is designed to recapitulate the 3D extracellular matrix of the trachea with stable mechanical properties imparted from the extraluminal airway splint. We performed segmental orthotopic tracheal replacement in a mouse microsurgical model. MicroCT was used to measure graft patency. Tracheal neotissue formation was quantified histologically. Airflow dynamic properties were analyzed using computational fluid dynamics. We found that CTG are easily implanted and did not result in vascular erosion, tracheal injury, or inflammation. Graft epithelialization and endothelialization were comparable with CTG to control. Tracheal collapse was absent with CTG. Composite tracheal scaffolds combine biocompatible synthetic support with PDTG, supporting the regeneration of host epithelium while maintaining graft structure.

Tracheal Macrophages During Regeneration and Repair of Long-Segment Airway Defects.

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, 132:737-746, 2022.

Modulation of Synthetic Tracheal Grafts with Extracellular Matrix Coatings.

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.

Regeneration of partially decellularized tracheal scaffolds in a mouse model of orthotopic tracheal replacement.

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.

Spatial and Temporal Analysis of Host Cells in Tracheal Graft Implantation.

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.

Deconstructing tissue engineered trachea: Assessing the role of synthetic scaffolds, segmental replacement and cell seeding on graft performance.

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.

Efficacy of exogenous pyruvate in TremblerJ mouse model of Charcot-Marie-Tooth neuropathy.

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.

AAV1.NT-3 gene therapy increases muscle fiber diameter through activation of mTOR pathway and metabolic remodeling in a CMT mouse model.

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.

Impaired regeneration in calpain-3 null muscle is associated with perturbations in mTORC1 signaling and defective mitochondrial biogenesis.

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-ß 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-ß 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.

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.

VIP-expressing dendritic cells protect against spontaneous autoimmune peripheral polyneuropathy.

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.

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.