Organotypic sinonasal airway culture systems are predictive of the mucociliary phenotype produced by bronchial airway epithelial cells.

Differentiated air-liquid interface models are the current standard to assess the mucociliary phenotype using clinically-derived samples in a controlled environment. However, obtaining basal progenitor airway epithelial cells (AEC) from the lungs is invasive and resource-intensive. Hence, we applied a tissue engineering approach to generate organotypic sinonasal AEC (nAEC) epithelia to determine whether they are predictive of bronchial AEC (bAEC) models.

Improved in-vivo airway gene transfer via magnetic-guidance, with protocol development informed by synchrotron imaging.

Gene vectors to treat cystic fibrosis lung disease should be targeted to the conducting airways, as peripheral lung transduction does not offer therapeutic benefit. Viral transduction efficiency is directly related to the vector residence time. However, delivered fluids such as gene vectors naturally spread to the alveoli during inspiration, and therapeutic particles of any form are rapidly cleared via mucociliary transit.

Single-Dose Lentiviral Mediated Gene Therapy Recovers CFTR Function in Cystic Fibrosis Knockout Rats.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator () gene, resulting in defective ion transport in the airways. Addition of a functioning 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.

Towards Human Translation of Lentiviral Airway Gene Delivery for Cystic Fibrosis: A One-Month and Reporter Gene Study in Marmosets.

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment.

An Whole-Organ Liver Engineering for Testing of Genetic Therapies.

Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics.

Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing.

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model.

Public Acceptability of Gene Therapy and Gene Editing for Human Use: A Systematic Review.

Gene therapy and gene editing technologies are complex and it can be difficult for the public to understand their possible benefits or side effects. However, patient and public support is critical for the successful adoption of any new technology. Given the recent advances in gene therapy and gene editing, their potential clinical benefits, and the significant attention that has been given to the first-known successful attempt at permanent and heritable changes to the human genome, a systematic review was performed to assess beliefs and attitudes toward gene therapy and gene editing for human use, and to highlight the factors that influence acceptability.

Continual Conscious Bioluminescent Imaging in Freely Moving Mice.

In vivo bioluminescent imaging allows the detection of reporter gene expression in rodents in real time. Here we describe a novel technology whereby we can generate somatotransgenic rodents with the use of a viral vector carrying a luciferase transgene. We are able to achieve long term luciferase expression by a single injection of lentiviral or adeno-associated virus vectors to newborn mice.

NF-κB Activity Initiates Human ESC-Derived Neural Progenitor Cell Differentiation by Inducing a Metabolic Maturation Program.

Human neural development begins at embryonic day 19 and marks the beginning of organogenesis. Neural stem cells in the neural tube undergo profound functional, morphological, and metabolic changes during neural specification, coordinated by a combination of exogenous and endogenous cues. The temporal cell signaling activities that mediate this process, during development and in the postnatal brain, are incompletely understood.

Gene Modified T Cell Therapies for Hematological Malignancies.

This article focuses on clinical applications of T cells transduced to express recombinant T cell receptor and chimeric antigen receptor constructs directed toward hematological malignancies, and considers newer strategies incorporating gene-editing technologies to address GvHD and host-mediated rejection. Recent data from clinical trials are reviewed, and an overview is provided of current and emerging manufacturing processes; consideration is also given to new developments in the pipeline.

Bioluminescence Monitoring of Promoter Activity In Vitro and In Vivo.

The application of luciferase reporter genes to provide quantitative outputs for the activation of promoters is a well-established technique in molecular biology. Luciferase catalyzes an enzymatic reaction, which in the presence of the substrate luciferin produces photons of light relative to its molar concentration. The luciferase transgene can be genetically inserted at the first intron of a target gene to act as a surrogate for the gene's endogenous expression in cells and transgenic mice.

Continual conscious bioluminescent imaging in freely moving somatotransgenic mice.

Luciferase bioimaging in living animals is increasingly being applied in many fields of biomedical research. Rodent imaging usually involves anaesthetising the animal during data capture, however, the biological consequences of anaesthesia have been largely overlooked. We have evaluated luciferase bioimaging in conscious, unrestrained mice after neonatal intracranial or intravascular administration of lentiviral, luciferase reporter cassettes (biosensors); we present real-time analyses from the first day of life to adulthood.

Genome-Edited T Cell Therapies.

Purpose Of Review: Alternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed.

Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice.

Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood.

BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity.

Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse + , but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of human alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming.

The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts.

In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood.

MicroRNA miR-107 is overexpressed in pituitary adenomas and inhibits the expression of aryl hydrocarbon receptor-interacting protein in vitro.

Abnormal microRNA (miRNA) expression profiles have recently been associated with sporadic pituitary adenomas, suggesting that miRNAs can contribute to tumor formation; miRNAs are small noncoding RNAs that inhibit posttranscriptional expression of target mRNAs by binding to target sequences usually located in the 3'-UTR. In this study, we investigated the role played by miR-107, a miRNA associated with different human cancers, in sporadic pituitary adenomas and its interaction with the pituitary tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP). miR-107 expression was evaluated in pituitary adenoma and normal pituitary samples using microRNA screen TLDA (TaqMan Low-Density Array) and RT-qPCR assays.

Choice of surrogate and physiological markers for prenatal gene therapy.

Surrogate genetically encoded markers have been utilized in order to analyze gene transfer efficacy, location, and persistence. These marker genes have greatly accelerated the development of gene transfer vectors for the ultimate application of gene therapy using therapeutic genes. They have also been used in many other applications, such as gene marking in order to study developmental cell lineages, to track cell migration, and to study tumor growth and metastasis.