The potential for gene therapy to be an effective treatment for cystic fibrosis (CF) airway disease has been limited by inefficient gene transfer vector particle delivery and lack of persistent gene expression. We have developed an airway conditioning process that, when combined with a human immunodeficiency virus (HIV)-derived lentivirus (LV) vector, resulted in persistent in vivo expression of transgenes in airway epithelium. Pretreatment of mouse nasal epithelium with the detergent lysophosphatidylcholine (LPC) prior to instillation of a single dose of an LV vector carrying the LacZ marker gene produced significant LacZ gene expression in nasal airway epithelium for at least 92 days. Transduction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene using the same LV vector system resulted in partial recovery of electrophysiologic function in the nasal airway epithelium of CF mice (cftrtm1Unc knockout) for at least 110 days. This first demonstration of LV-mediated in vivo recovery of CFTR function in CF airway epithelium illustrates the potential of combining a preconditioning of the airway surface with a simple and brief LV vector exposure to produce therapeutic gene expression in airway.
Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene, resulting in defective ion transport in the airways. Addition of a functioning CFTR gene into affected airway cells has the potential to be an effective treatment for lung disease. The therapeutic efficacy of airway gene transfer can be quantified in animal models by assessing ion transport in the treated nasal epithelium using the nasal potential difference (PD) measurement technique. The nasal PD technique is routinely used in CF mice, however when applied to a recently developed CF rat model those animals did not tolerate the initial nasal PD assessment, therefore the procedure was firstly optimised in rats. This study evaluated the effect of lentiviral (LV)-mediated CFTR airway gene delivery on nasal PD in a CFTR knockout rat model. LV gene vector containing the CFTR gene tagged with a V5 epitope tag (LV-V5- CFTR ) was delivered to the nasal epithelium of CF rats, and one week later nasal PD was analysed. This study demonstrated for the first time that LV-V5- CFTR treatment produced a mean correction of 46% towards wild-type chloride response in treated CF rats. Transduced cells were subsequently identifiable using V5 immunohistochemical staining. These findings in the nose validate the use of airway gene therapy for future lung based experiments.
Abstract Computed tomography (CT) and spirometry are the mainstays of clinical pulmonary assessment. Spirometry is effort dependent and only provides a single global measure that is insensitive for regional disease and as such, poor for capturing the early onset of lung disease, especially patchy disease such as cystic fibrosis lung disease. CT sensitively measures change in structure associated with advanced lung disease. However, obstructions in the peripheral airways and early onset of lung stiffening are often difficult to detect. Furthermore, CT imaging poses a radiation risk, particularly for young children and dose reduction tends to result in reduced resolution. Here, we apply a series of lung tissue motion analyses, to achieve regional pulmonary function assessment in β-ENaC-overexpressing mice, a well-established model of lung disease. The expiratory time constants of regional airflows in the segmented airway tree were quantified as a measure of regional lung function. Our results showed marked heterogeneous lung function in β-ENaC-Tg mice compared to wild-type littermate controls; identified locations of airway obstruction and quantified regions of bimodal airway resistance demonstrating lung compensation. These results demonstrate the applicability of regional lung function derived from lung motion as an effective alternative respiratory diagnostic tool.
Single-cell RNA sequencing (scRNA-seq) studies identified a novel subpopulation of epithelial cells along the rostrocaudal axis of human intestine specifically marked by bestrophin 4 (BEST4) that are enriched for genes regulating pH, GPCR acid-sensing receptors, satiety, cGMP signaling, HCO3- secretion, ion transport, neuropeptides, and paracrine hormones. Interestingly, BEST4+ cells in the proximal small intestine express CFTR but have not been linked to the previously described CFTR High Expresser Cell (CHE) subpopulation in rat and human intestine. ScRNA-seq studies in rat jejunum identified CHEs and a gene expression profile consistent with human small intestinal BEST4+ and neuropod cells. Protein immunolocalization confirmed that CHEs express CFTR, BEST4, neuropod proteins, high levels of intracellular uroguanylin (UGN), guanylyl cyclase-C (GC-C), and the proton channel otopetrin 2 (OTOP2), and display long basal processes connecting to neurons. OTOP2, GC-C, and CFTR traffic robustly into the apical domain of CHEs in response to acidic luminal conditions, indicating their roles in luminal pH regulation. In the ΔF508 cystic fibrosis (CF) rat jejunum, the loss of apical CFTR did not affect BEST4 protein expression in CHEs. However, there was an increased abundance of CHE cells in the ΔF508 rat jejunum compared to wild-type animals. Furthermore, ΔF508 rat CHEs expressed higher levels of GC-C at the apical domain compared to wild-type. These data implicate CHEs in intestinal CF disease pathogenesis.
Abstract Objectives Cobb angle is a standard method for quantification of scoliosis in adolescent idiopathic scoliosis to guide treatment decisions. Precise and timely curve detection can ensure early referrals, amenable for bracing. Radiology reports serve as a guiding tool for family physicians to expedite specialist referrals. Therefore, accurate and reliable measurement of Cobb angle at the community level is crucial. This retrospective study investigated the agreement in Cobb angle measurement between radiologists and spine surgeons. Methods Eighty radiographic reports (Cobb angle, Risser stage, and end vertebrae selection) completed by radiologists and spine surgeons were compared. To assess interrater reliability, interclass correlation coefficients (ICC) with 95% confidence intervals (CIs) were computed. ICC < 0.70, 0.70 to 0.79, 0.80 to 89, and 0.9 to 0.99 were considered poor, fair, good, and excellent reliability, respectively. All radiographs were assessed for quality. Results The agreement between spine surgeons and radiologists was poor (ICC = 0.65, 95% CI: 0.13 to 0.97). The agreement between spine surgeons and community radiologists was poor (ICC = 0.45, 95% CI: 0.17 to 0.66). Risser stage was not reported in 56 of the 80 reports. ICC between spine surgeons and radiologists for the Risser stage was poor (ICC = 0.625, 95% CI: 0.325 to 0.794). For end vertebrae identification, there was absolute agreement of end vertebrae identification in 23 of the 80 scans. Conclusions This study demonstrated a significant disagreement in scoliosis measurement between radiologists and spine surgeons, which significantly impacts appropriateness of referrals. Methods to improve triaging using allied health professional (i.e., nurse practitioners) may help ensure that patients presenting with scoliosis are referred in a timely manner.
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