High-throughput transcriptome analyses from ASPIRO, a phase 1/2/3 study of gene replacement therapy for X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a severe congenital disease characterized by profound muscle weakness, respiratory failure, and early death. No approved therapy for XLMTM is currently available. Adeno-associated virus (AAV)-mediated gene replacement therapy has shown promise as an investigational therapeutic strategy. We aimed to characterize the transcriptomic changes in muscle biopsies of individuals with XLMTM who received resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) in the ASPIRO clinical trial and to identify potential biomarkers that correlate with therapeutic outcome. We leveraged RNA-sequencing data from the muscle biopsies of 15 study participants and applied differential expression analysis, gene co-expression analysis, and machine learning to characterize the transcriptomic changes at baseline (pre-dose) and at 24 and 48 weeks after resamirigene bilparvovec dosing. As expected, MTM1 expression levels were significantly increased after dosing (p < 0.0001). Differential expression analysis identified upregulated genes after dosing that were enriched in several pathways, including lipid metabolism and inflammatory response pathways, and downregulated genes were enriched in cell-cell adhesion and muscle development pathways. Genes involved in inflammatory and immune pathways were differentially expressed between participants exhibiting ventilator support reduction of either greater or less than 6 h/day after gene therapy compared to pre-dosing. Co-expression analysis identified similarly regulated genes, which were grouped into modules. Finally, the machine learning model identified five genes, including MTM1, as potential RNA biomarkers to monitor the progress of AAV gene replacement therapy. These findings further extend our understanding of AAV-mediated gene therapy in individuals with XLMTM at the transcriptomic level.

Effect of HDAC Inhibitors on Corneal Keratocyte Mechanical Phenotypes in 3-D Collagen Matrices.

PURPOSE: Histone deacetylase inhibitors (HDAC) have been shown to inhibit the TGFß-induced myofibroblast transformation of corneal fibroblasts in 2-D culture. However, the effect of HDAC inhibitors on keratocyte spreading, contraction, and matrix remodeling in 3-D culture has not been directly assessed. The goal of this study was to investigate the effects of the HDAC inhibitors Trichostatin A (TSA) and Vorinostat (SAHA) on corneal keratocyte mechanical phenotypes in 3-D culture using defined serum-free culture conditions. METHODS: Rabbit corneal keratocytes were plated within standard rat tail type I collagen matrices (2.5 mg/ml) or compressed collagen matrices (~100 mg/ml) and cultured for up to 4 days in serum-free media, PDGF BB, TGFß1, and either 50 nM TSA, 10 µM SAHA, or vehicle (DMSO). F-actin, α-SM-actin, and collagen fibrils were imaged using confocal microscopy. Cell morphology and global matrix contraction were quantified digitally. The expression of α-SM-actin was assessed using western blotting. RESULTS: Corneal keratocytes in 3-D matrices had a quiescent mechanical phenotype, as indicated by a dendritic morphology, a lack of stress fibers, and minimal cell-induced matrix remodeling. This phenotype was generally maintained following the addition of TSA or SAHA. TGFß1 induced a contractile phenotype, as indicated by a loss of dendritic cell processes, the development of stress fibers, and significant matrix compaction. In contrast, cells cultured in TGFß1 plus TSA or SAHA remained dendritic and did not form stress fibers or induce ECM compaction. Western blotting showed that the expression of α-SM actin after treatment with TGFß1 was inhibited by TSA and SAHA. PDGF BB stimulated the elongation of keratocytes and the extension of dendritic processes within 3-D matrices without inducing stress fiber formation or collagen reorganization. This spreading response was maintained in the presence of TSA or SAHA. CONCLUSIONS: Overall, HDAC inhibitors appear to mitigate the effects of TGFß1 on the transformation of corneal keratocytes to a contractile, myofibroblast phenotype in both compliant and rigid 3-D matrices while preserving normal cell spreading and their ability to respond to the pro-migratory growth factor PDGF.

Correction: Petroll et al. Experimental Models for Investigating Intra-Stromal Migration of Corneal Keratocytes, Fibroblasts and Myofibroblasts. J. Funct. Biomater. 2012, 3, 183-198.

In the original publication [...].

Corneal stromal cells use both high- and low-contractility migration mechanisms in 3-D collagen matrices.

Corneal keratocyte migration can impact both corneal clarity and refractive outcome following injury or refractive surgery. In this study, we investigated how culture conditions, ECM properties, and Rho kinase activity regulate the mechanics of keratocyte migration, using a nested collagen matrix model. Time-lapse imaging demonstrated that both serum and PDGF stimulate keratocyte migration into the outer matrix. Although the velocity of cell migration was similar, cells in serum were bipolar and induced significant matrix deformation during migration, whereas PDGF induced extension of branching dendritic processes with smaller, more localized force generation. These differences in cell-induced matrix reorganization were verified with a global matrix contraction assay and confocal reflection imaging, using both bovine and rat tail collagen. When constructs were detached from the substrate to lower the effective stiffness, migration was significantly reduced in serum; but was unchanged in PDGF. These differences in migration mechanics were mediated, in part, by Rho kinase. Overall, corneal keratocytes can effectively migrate through collagen matrices using varying degrees of cellular force generation. Low-contractility migration may facilitate keratocyte repopulation of the stroma following surgery or injury, without altering the structural and mechanical properties that are critical to maintaining corneal transparency.

Characterization of corneal keratocyte morphology and mechanical activity within 3-D collagen matrices.

The purpose of this study was to assess quantitatively the differences in morphology, cytoskeletal organization and mechanical behavior between quiescent corneal keratocytes and activated fibroblasts in a 3-D culture model. Primary cultures of rabbit corneal keratocytes and fibroblasts were plated inside type I collagen matrices in serum-free media or 10% FBS, and allowed to spread for 1-5 days. Following F-actin labeling using phalloidin, and immunolabeling of tubulin, alpha-smooth muscle actin or connexin 43, fluorescent and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, dynamic imaging was performed using differential interference contrast microscopy, and finite element modeling was used to map ECM deformations. Corneal keratocytes developed a stellate morphology with numerous cell processes that ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. Fibroblasts on the other hand, had a more bipolar morphology with pseudopodial processes (P

An experimental model for assessing fibroblast migration in 3-D collagen matrices.

The purpose of this study was to develop and test a novel culture model for studying fibroblast migration in 3-D collagen matrices. Human corneal fibroblasts were seeded within dense, randomly oriented compressed collagen matrices. A 6 mm diameter button of this cell-seeded matrix was placed in the middle of an acellular, less dense outer collagen matrix. These constructs were cultured for 1, 3, 5 or 7 days in serum-free media, 10% fetal bovine serum (FBS), or 50 ng/ml PDGF. Constructs were then fixed and labeled with AlexaFluor 546 phalloidin (for f-actin) and TOTO-3 (for nuclei). Cell-matrix interactions were assessed using a combination of fluorescent and reflected light confocal imaging. Human corneal fibroblasts in serum-free media showed minimal migration into the outer (non-compressed) matrix. In contrast, culture in serum or PDGF stimulated cell migration. Cell-induced collagen matrix reorganization in the outer matrix could be directly visualized using reflected light imaging, and was highest following culture in 10% FBS. Cellular contraction in 10% FBS often led to alignment of cells parallel to the outer edge of the inner matrix, similar to the pattern observed during corneal wound healing following incisional surgery. Overall, this 3-D model allows the effects of different culture conditions on cell migration and matrix remodeling to be assessed simultaneously. In addition, the design allows for ECM density, geometry and mechanical constraints to be varied in a controlled fashion. These initial results demonstrate differences in cell and matrix patterning during migration in response to serum and PDGF.

Regulation of corneal fibroblast morphology and collagen reorganization by extracellular matrix mechanical properties.

PURPOSE: To investigate how extracellular matrix mechanical properties influence cell and matrix patterning in three-dimensional culture. METHODS: Human corneal fibroblasts were seeded within 30 x 10 mm collagen matrices that were unconstrained (UN), fully constrained (CO) along the long axis by attaching the construct to two immobilized plastic bars, or partially constrained (PC) by allowing linear elastic displacement of one bar. After 24 hours, constructs were labeled with phalloidin and were imaged using fluorescent and reflected light (for collagen) confocal microscopy. Cell morphology and local collagen fibril density and alignment were measured using digital image processing. RESULTS: Corneal fibroblasts in UN matrices were less elongated (UN < PC < CO; P < 0.05) than those in constrained matrices. Cells were aligned parallel to the long axis in the anisotropic region of constrained matrices but were randomly aligned in unconstrained (isotropic) matrices (UN < PC = CO; P < 0.05). Both the local collagen density and the degree of cell/collagen coalignment were higher in constrained matrices (UN < PC < CO; P < 0.05). In regions of higher cell density, additional bands of aligned collagen were often observed between individual cells. CONCLUSIONS: These data suggest that cell spreading, alignment, and contractile force generation are directly influenced by the mechanical properties of the surrounding extracellular matrix (ECM). Corneal fibroblasts generally align and compact collagen parallel to the axis of greatest ECM stiffness. Mechanical cross-talk between adjacent cells leads to enhancement of matrix reorganization, and results in additional, more complex matrix patterning.

Assessment of corneal epithelial integrity after acute exposure to ocular hypotensive agents preserved with and without benzalkonium chloride.

The corneal toxicity of 2 intraocular pressure-lowering agents was compared in a rabbit cornea model with New Zealand White rabbits. Corneal epithelial morphology and cell size were assessed by in vivo confocal microscopy. Baseline microscopic examinations were performed on 1 eye of each animal. Two weeks later, the eyes were bathed for 3 min in travoprost 0.004% preserved without benzalkonium chloride (BAK( or latanoprost 0.005% preserved with 0.02% BAK; the eyes were then rinsed with balanced salt solution, and the corneas were again examined by confocal microscopy (n=4/group). A second group of animals was exposed to the medications through a dosing regimen of 1 drop/min (lpar3 drops total) (n=4/group). In eyes treated with travoprost without BAK (3-min bath), superficial epithelial cells were similar to baseline, as indicated by their visible cell borders and bright nuclei. In contrast, the surface cells in eyes treated with latanoprost were significantly smaller and brighter and had less distinct borders. Surface cell size was significantly smaller as compared with baseline size and as compared with rabbits treated with travoprost without BAK for 3 min. Similar effects on corneal epithelial cell morphology were observed with the 1-drop/min dosing regimen. In this rabbit model, travoprost 0.004% preserved without BAK did not cause corneal epithelial toxicity; latanoprost 0.005% induced superficial cell loss, most likely caused by the presence of a relatively high concentration of BAK (0.02%).

Fibroblastic Transformation of Corneal Keratocytes by Rac Inhibition is Modulated by Extracellular Matrix Structure and Stiffness.

The goal of this study was to investigate how alterations in extracellular matrix (ECM) biophysical properties modulate corneal keratocyte phenotypes in response to specific wound healing cytokines and Rho GTPases. Rabbit corneal keratocytes were plated within standard collagen matrices (2.5 mg/mL) or compressed collagen matrices (~100 mg/mL) and cultured in serum-free media, PDGF BB, IGF, FGF2 or TGFß1, with or without the Rac1 inhibitor NSC23766 and/or the Rho kinase inhibitor Y-27632. After 1 to 4 days, cells were labeled for F-actin and imaged using confocal microscopy. Keratocytes within standard collagen matrices (which are highly compliant) maintained a dendritic phenotype following culture in serum-free media, PDGF, IGF and FGF, but developed stress fibers in TGFß1. Keratocytes within compressed collagen (which has high stiffness and low porosity) maintained a dendritic phenotype following culture in serum-free media, PDGF and IGF, but developed stress fibers in both FGF and TGFß1. The Rac inhibitor had no significant impact on growth factor responses in compliant matrices. Within compressed collagen matrices however, the Rac inhibitor induced fibroblastic transformation in serum-free media, PDGF and IGF. Fibroblast and myofibroblast transformation was blocked by Rho kinase inhibition. Overall, keratocyte growth factor responses appear to be regulated by both the interplay between Rho and Rac signaling, and the structural and mechanical properties of the ECM.

Growth factor regulation of corneal keratocyte mechanical phenotypes in 3-D collagen matrices.

PURPOSE: To assess how wound healing cytokines and the extracellular matrix (ECM) environment regulate the keratocyte mechanical phenotype. METHODS: Rabbit corneal keratocytes were plated within standard bovine or rat tail type I collagen matrices (2.5 mg/mL), compressed collagen matrices (approximately 100 mg/mL), or on collagen-coated dishes and cultured for up to 7 days in serum-free media, platelet derived growth factor BB (PDGF BB), insulin-like growth factor (IGF), TGFß1, TGFß2, or FGF2. F-actin, α-smooth muscle actin (α-SMA) and collagen fibrils were imaged using confocal microscopy. Cell morphology, local matrix reorganization, and global matrix contraction were quantified digitally. RESULTS: IGF and PDGF BB stimulated elongation of keratocytes and extension of dendritic processes within 3-D matrices, without inducing stress fiber formation or collagen reorganization. In contrast, treatment with TGFß1 and TGFß2 increased keratocyte contractility, as indicated by stress fiber formation and matrix compaction and alignment. This transformation was enhanced at higher cell densities within standard 3-D matrices, in which α-SMA was incorporated into stress fibers. In contrast, α-SMA was expressed within compressed 3-D matrices even at low cell density. FGF2 did not produce significant cytoskeletal or matrix reorganization in standard 3-D matrices; however, stress fibers were consistently expressed within compressed collagen matrices and on rigid two-dimensional substrates. Inhibiting Rho kinase blocked both TGFß- and FGF2-induced stress fiber formation. CONCLUSIONS: Keratocytes cultured in IGF or PDGF BB maintain a quiescent mechanical phenotype over a range of matrix environments. In contrast, the mechanical phenotypes induced by FGF and TGFß vary in response to the structural and/or mechanical properties of the ECM.