Assessment of Lentiviral Vector Mediated CFTR Correction in Mice Using an Improved Rapid in vivo Nasal Potential Difference Measurement Protocol.

Cystic Fibrosis (CF) is caused by a defect in the CF transmembrane conductance regulator (CFTR) gene responsible for epithelial ion transport. Nasal potential difference (PD) measurement is a well established diagnostic technique for assessing the efficacy of therapies in CF patients and animal models. The aim was to establish a rapid nasal PD protocol in mice and quantify the efficacy of lentiviral (LV) vector-based CFTR gene therapy. Anaesthetised wild-type (WT) and CF mice were non-surgically intubated and nasal PD measurements were made using a range of buffer flow rates. Addition of the cAMP agonist, isoproterenol, to the buffer sequence was then examined. The optimised rapid PD technique was then used to assess CFTR function produced by second and third generation LV-CFTR vectors. V5 epitope tagged-CFTR in nasal tissue was identified by immunohistochemistry. When intubated, mice tolerated higher flow rates. Isoproterenol could discriminate between WT and CF mice. Improved chloride transport was observed for the second and third generation LV-CFTR vectors, with up to 60% correction of the cAMP-driven chloride response towards WT. V5-CFTR was located in ciliated epithelial cells. The rapid PD technique enables improved functional assessment of the bioelectrical ion transport defect for both current and potential CF therapies.

Ibrexafungerp: A novel oral glucan synthase inhibitor.

Ibrexafungerp is a novel glucan synthase inhibitor currently undergoing phase II and phase III clinical trials. This compound has demonstrated in vitro activity against clinically important fungal pathogens including Candida spp. and Aspergillus spp. It is able to retain activity against many echinocandin-resistant strains of Candida due to differential avidity for the target site compared to echinocandins. In vivo animal models have demonstrated efficacy in murine models of invasive candidiasis, aspergillosis, and pneumocystis. Due to high bioavailability, it can be administered both orally and intravenously. A favorable drug interaction and tolerability profile is observed with this compound. This review summarizes existing data that have either been published or presented at international symposia.

Airway gene transfer in a non-human primate: lentiviral gene expression in marmoset lungs.

Genetic therapies for cystic fibrosis (CF) must be assessed for safety and efficacy, so testing in a non-human primate (NHP) model is invaluable. In this pilot study we determined if the conducting airways of marmosets (n = 2) could be transduced using an airway pre-treatment followed by an intratracheal bolus dose of a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector (LacZ reporter). LacZ gene expression (X-gal) was assessed after 7 days and found primarily in conducting airway epithelia as well as in alveolar regions. The LacZ gene was not detected in liver or spleen via qPCR. Vector p24 protein bio-distribution into blood was transient. Dosing was well tolerated. This preliminary study confirmed the transducibility of CF-relevant airway cell types. The marmoset is a promising NHP model for testing and translating genetic treatments for CF airway disease towards clinical trials.

Synchrotron phase-contrast X-ray imaging reveals fluid dosing dynamics for gene transfer into mouse airways.

Although airway gene transfer research in mouse models relies on bolus fluid dosing into the nose or trachea, the dynamics and immediate fate of delivered gene transfer agents are poorly understood. In particular, this is because there are no in vivo methods able to accurately visualize the movement of fluid in small airways of intact animals. Using synchrotron phase-contrast X-ray imaging, we show that the fate of surrogate fluid doses delivered into live mouse airways can now be accurately and non-invasively monitored with high spatial and temporal resolution. This new imaging approach can help explain the non-homogenous distributions of gene expression observed in nasal airway gene transfer studies, suggests that substantial dose losses may occur at deliver into mouse trachea via immediate retrograde fluid motion and shows the influence of the speed of bolus delivery on the relative targeting of conducting and deeper lung airways. These findings provide insight into some of the factors that can influence gene expression in vivo, and this method provides a new approach to documenting and analyzing dose delivery in small-animal models.

Laser fluorescence bronchoscopy for detection of fluorescent reporter genes in airway epithelia.

Current methods for detecting successful gene transfer to airway epithelia involve obtaining a sample of the target tissue. This may affect the longevity of expression of the transgene under evaluation. We describe a laser fluorescence bronchoscopic system that can detect the expression of the fluorescent protein, green fluorescence protein (GFP), in the airway of monkeys that have been transfected with adenovirus, without the need for obtaining tissue. This technique will have applications in pre-clinical and clinical studies of gene transfer to airway epithelia and other surface epithelia accessible by endoscopy.

Macular electroretinograms: their accuracy, specificity and implementation for clinical use.

The pattern and the focal electroretinogram (ERG) are both non-invasive, electrophysiological responses recorded from circumscribed retinal areas and are most easily recorded from the macula. This paper describes how our department has incorporated these tests into our clinical protocol, shows how the recording technique and the method of electrode construction may be improved, and describes the normal limits of the macular responses we obtain. The ERG signal-noise ratio we obtained was better than that of the binocular visual evoked potentials (VEPs) recorded simultaneously. Pattern and focal ERGs, using improved methods of recording, show promise of being a valuable addition to the clinical investigation of subtle maculopathies and some forms of optic nerve dysfunction. Three illustrative cases are described. The first demonstrates normal macular ERG responses with abnormal Ganzfeld ERGs due to peripheral retinal damage. The second reveals differential pattern ERG reduction with normal focal ERG in recent optic neuritis. The third case demonstrates reversible simultaneous loss of Ganzfeld ERGs and macular ERGs in vitamin A deficiency.