Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening.

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient's immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and ß-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures.

BACKGROUND: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. OBJECTIVE: To compare methods currently used to quantify antisense oligonucleotide-induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. METHODS: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. RESULTS: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. CONCLUSIONS: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR.

Acute and sustained effects of aerosolized vs. bolus surfactant therapy in premature lambs with respiratory distress syndrome.

BACKGROUND: Surfactant (SF) instillation may produce acute deleterious effects on gas exchange and both systemic and cerebral hemodynamics. Our aim was to compare the effects of aerosolized SF (SF-aero) with those of bolus SF (SF-bolus) administration on gas exchange, lung mechanics, and cardiovascular function in premature lambs with respiratory distress syndrome (RDS). METHODS: Fourteen preterm lambs (85% gestation) were randomly assigned to receive SF-aero or SF-bolus. Oxygenation index (OI), PaCO2, cardiovascular parameters, carotid blood flow (CBF), lung compliance (mean dynamic compliance), and tidal volume (VT) were measured every 30 min for 6 h. Biochemical and histological analyses were performed. RESULTS: After delivery, lambs developed severe RDS (inspiratory fraction of oxygen: 1; pH < 7.15; PaCO2 > 80 mm Hg; PaO2 < 30 mm Hg, mean dynamic compliance < 0.08 ml/cm H2O/kg). By 60 min after treatment, both groups showed an improvement in OI, PaCO2, mean dynamic compliance, and VT that was maintained until the end of the experiment. PaCO2 and CBF increased significantly in the SF-bolus group during the first 15-30 min, without concomitant changes in cardiovascular parameters, whereas in the SF-aero group, PaCO2 and CBF decreased gradually. SF-aero induced less alveolar hemorrhage and inflammation. CONCLUSION: SF-aero produced improvements in gas exchange and lung mechanics similar to those produced by bolus administration but with less lung injury and fewer cerebral hemodynamic changes.

Aerosolized perfluorocarbon improves gas exchange and pulmonary mechanics in preterm lambs with severe respiratory distress syndrome.

BACKGROUND: Aerosolized perfluorocarbon (PFC) has been proposed as an alternative method of PFC administration; however, the efficacy of aerosolized PFC in a preterm animal model has not yet been demonstrated. METHODS: Twelve preterm lambs were randomized to two groups: a perfluorodecalin (PFD) aerosol group (n = 6) receiving 10 ml/kg/h of PFD delivered by an intratracheal inhalation catheter followed by 4 h of mechanical ventilation (MV) or the control group, in which animals (n = 6) were managed for 6 h with MV. Gas exchange, pulmonary mechanics, cardiovascular parameters, and cerebral blood flow (CBF) were measured. RESULTS: Both groups developed hypoxia, hypercarbia, and acidosis at baseline. Aerosolized PFD improved oxygenation (P < 0.0001) and pulmonary mechanics (P < 0.0001) and changed carbon dioxide values to normal physiological levels, unlike the treatment given to the controls (P < 0.0003). The time course of mean arterial blood pressure and CBF were significantly affected by PFD aerosolization, especially during the first hour of life. CBF gradually decreased during the first hour in the PFD aerosol group and remained stable until the end of the follow-up, whereas CBF remained higher in the control group (P < 0.0028). CONCLUSION: Aerosolized PFD improves pulmonary function in preterm lambs and should be further investigated as an alternative mode of PFC administration.

Surfactant and perfluorocarbon aerosolization during different mechanical ventilation strategies by means of inhalation catheters: an in vitro study.

BACKGROUND: Aerosol delivery of surfactant and perfluorocarbon (PFC) is a desirable therapeutic approach for the treatment of various lung diseases in patients undergoing mechanical ventilation. However, the behavior of these substances during aerosolization differs significantly from that of aqueous solutions. In particular, the high vapor pressure of many PFCs tends to result in greater evaporation during mechanical ventilation. METHODS: Three PFCs and surfactant were aerosolized during mechanical ventilation by means of three intratracheal inhalation catheters (IC) with different air flow rates (IC-1.23, IC-1.1, and IC-1.4), with their aerosol generating tip placed at the distal end of the endotracheal tube (i.d. 4 mm). The influence of four different ventilation strategies on aerosol production rate and PFC and surfactant recovery was studied. The changes in intrapulmonary pressure produced by the air jets of each IC were measured. RESULTS: With IC-1.23 and IC-1.1, the highest rates of aerosol production were achieved using FC75 (2.27±0.18 and 0.76±0.01, respectively) followed by PFOB (1.74±0.06 and 0.56±0.04), PFD (0.82±0.01 and 0.21±0.01), and surfactant (0.42±0.05 and 0.092±0.01). With IC-1.4 modest aerosol production was obtained irrespective of the aerosolized compound. Mechanical ventilation influenced aerosol recovery, with the trend being toward recovering higher percentages of the compounds with lower peak inspiratory pressure (PIP) and lower respiratory rate (RR) settings. The highest percentages of the initial volume were recovered with IC-1.23 (between 65.43%±4.2 FC75 and 90.21%±4.71 surfactant) followed by IC-1.1 (between 46.48%±4.46 FC75 and 73.19%±2.82 PFOB) and IC-1.4 (between 4.65%±4.36 FC75 and 63.24%±9.71 surfactant). Each of three of the ICs were found to increase the intrapulmonary pressure by about 2-3 cmH2O during mechanical ventilation. CONCLUSIONS: Despite of mechanical ventilation, IC-1.23 and IC-1.1 were able to deliver significant amounts of surfactant and perfluorocarbon to the lung model. Changes in PIP and RR directly influence the percentage of surfactant and perfluorocarbon recovered.

Early Cerebral Hemodynamic, Metabolic, and Histological Changes in Hypoxic-Ischemic Fetal Lambs during Postnatal Life.

The hemodynamic, metabolic, and biochemical changes produced during the transition from fetal to neonatal life may be aggravated if an episode of asphyxia occurs during fetal life. The aim of the study was to examine regional cerebral blood flow (RCBF), histological changes, and cerebral brain metabolism in preterm lambs, and to analyze the role of oxidative stress in the first hours of postnatal life following severe fetal asphyxia. Eighteen chronically instrumented newborn lambs were randomly assigned to either a control group or the hypoxic-ischemic (HI) group, in which case fetal asphyxia was induced just before delivery. All the animals were maintained on intermittent positive pressure ventilation for 3 h after delivery. During the HI insult, the injured group developed acidosis, hypoxia, hypercapnia, lactic acidosis, and tachycardia (relative to the control group), without hypotension. The intermittent positive pressure ventilation transiently improved gas exchange and cardiovascular parameters. After HI injury and during ventilatory support, there continued to be an increased RCBF in inner regions among the HI group, but no significant differences were detected in cortical flow compared to the control group. Also, the magnitude of the increase in TUNEL positive cells (apoptosis) and antioxidant enzymes, and decrease of ATP reserves was significantly greater in the brain regions where the RCBF was not higher. In conclusion, our findings identify early metabolic, histological, and hemodynamic changes involved in brain damage in premature asphyxiated lambs. Such changes have been described in human neonates, so our model could be useful to test the safety and the effectiveness of different neuroprotective or ventilation strategies applied in the first hours after fetal HI injury.