Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy.

OBJECTIVE: To report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping. METHODS: Part 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry. RESULTS: Twelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%-4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry. CONCLUSION: Golodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization. CLINICALTRIALSGOV IDENTIFIER: NCT02310906. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.

Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.

Validation of a Muscle-Specific Tissue Image Analysis Tool for Quantitative Assessment of Dystrophin Staining in Frozen Muscle Biopsies.

CONTEXT.­: Duchenne muscular dystrophy is a rare, progressive, and fatal neuromuscular disease caused by dystrophin protein loss. Common investigational treatment approaches aim at increasing dystrophin expression in diseased muscle. Some clinical trials include assessments of novel dystrophin production as a surrogate biomarker of efficacy, which may predict a clinical benefit from treatment. OBJECTIVES.­: To establish an immunofluorescent scanning and digital image analysis workflow that provides an objective approach for staining intensity assessment of the immunofluorescence dystrophin labeling and determination of the percentage of biomarker-positive fibers in muscle cryosections. DESIGN.­: Optimal and repeatable digital image capture was achieved by a rigorously qualified fluorescent scanning process. After scanning qualification, the MuscleMap (Flagship Biosciences, Westminster, Colorado) algorithm was validated by comparing high-power microscopic field total and dystrophin-positive fiber counts obtained by trained pathologists to data derived by MuscleMap. Next, the algorithm was tested on whole-slide images of immunofluorescent-labeled muscle sections from Duchenne muscular dystrophy, Becker muscular dystrophy, and control patients. RESULTS.­: When used under the guidance of a trained pathologist, the digital image analysis tool met predefined validation criteria and demonstrated functional and statistical equivalence with manual assessment. This work is the first, to our knowledge, to qualify and validate immunofluorescent scanning and digital tissue image-analysis workflow, respectively, with the rigor required to support the clinical trial environments. CONCLUSIONS.­: MuscleMap enables analysis of all fibers within an entire muscle biopsy section and provides data on a fiber-by-fiber basis. This will allow future clinical trials to objectively investigate myofibers' dystrophin expression at a greater level of consistency and detail.

Low-level dystrophin expression attenuating the dystrophinopathy phenotype.

The reading frame rule suggests that Duchenne muscular dystrophy (DMD) results from DMD mutations causing an out-of-frame transcript, whereas the milder Becker muscular dystrophy results from mutations causing an in-frame transcript. However, predicted nonsense mutations may instead result in altered splicing and an in-frame transcript. Here we report a 10-year-old boy with a predicted nonsense mutation in exon 42 who had a 6-minute walk time of 157% of that of age matched DMD controls, characterized as intermediate muscular dystrophy. RNA sequencing analysis from a muscle biopsy revealed only 6.0-9.8% of DMD transcripts were in-frame, excluding exon 42, and immunoblot demonstrated only 3.2% dystrophin protein expression. Another potential genetic modifier noted was homozygosity for the protective IAAM LTBP4 haplotype. This case suggests that very low levels of DMD exon skipping and dystrophin protein expression may result in amelioration of skeletal muscle weakness, a finding relevant to current dystrophin-restoring therapies.

Basal prostate epithelial cells stimulate the migration of prostate cancer cells.

Carcinoma cells in PIN are situated above a layer of basal epithelial cells, which shield the tumor cells from stimulation by factors from the prostate stroma. During progression to invasive carcinoma, the basal cell layer becomes disrupted and tumor cells adhere to the basement membrane. The close proximity of basal epithelial cells to tumor cells in the early stages of prostate oncogenesis raises the possibility that basal epithelial cells participate in tumor cell invasion. Here, we investigated the migration-promoting activity of secreted factors from basal epithelial cells on BPH-1 cells, which we used as an in vitro model of preinvasive prostate cancer cells. We showed that the conditioned medium of basal epithelial cells (PEC-CM) contains adhesion proteins and chemotactic factors that stimulate adhesion, planar polarization, migration, and phosphorylation of Akt and that LY294002 and Wortmannin partially inhibit PEC-CM-triggered migration. We identified laminin-5 as a major migration-stimulating protein for BPH-1 cells in PEC-CM. Laminin-5 induced migration is completely inhibited by LY294002 or Wortmannin. In addition, antibody-depletion of laminin-5 from PEC-CM significantly diminishes the migration of BPH-1 cells. These results demonstrated, that laminin-5 is secreted by basal prostate epithelial cells in vivo and in vitro and stimulates migration of BPH-1 cells through a PI3-kinase dependent mechanism. Altogether, the possibility that basal epithelial cells assist in the invasion of in situ carcinoma cells is supported by the results from our in vitro system.

Laminin 5 deposition regulates keratinocyte polarization and persistent migration.

Repair of wounded epidermis requires both keratinocyte migration and deposition of laminin 5 over exposed dermal collagen. To understand the coupling between leading cell migration and laminin 5 deposition, we developed a novel migration assay using time-lapse microscopy. We demonstrate that in migrating, human keratinocytes the deposition of laminin 5 promoted 'processive migration', characterized by stable cell polarization that was tightly coupled to persistent, linear migration in the absence of a chemotactic gradient. Processive migration required deposition of laminin 5, which was restricted to the rear of the polar cell. Integrin alpha 3 beta 1 interacted with these laminin 5 deposits at contact sites that did not require actin-dependent cross-linking. Further, we show that the migrating cells switched adhesion by integrin alpha 2 beta 1 on collagen at the front of the cell to integrin alpha 3 beta 1 on exogenous laminin 5 at the rear of the cell. Along with this switch of integrin usage was the removal of collagen from sites under the cell that precisely correlated with deposition of laminin 5. Processive migration was blocked with suppressors of microtubule dynamics (nocodazole and taxol) or rottlerin, a PKC-delta inhibitor. These drugs were also shown to block deposition of laminin 5 but, surprisingly, constitutive secretion was unimpaired, suggesting deposition was a regulated event. Thus, at the front of the cell, the leading lamellipodium was stabilized through integrin interactions in focal complexes with the exogenous substratum. However, at the rear of the cell, stable cell polarization and linear migration was promoted by laminin 5 deposits and integrin alpha 3 beta 1.