Cystic fibrosis is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. While cystic fibrosis is a multi-organ disease, the leading causes of morbidity and mortality are related to progressive lung disease. Current understanding of the effects of the broad spectrum of CFTR mutations on CFTR function has allowed for the development of CFTR modulator therapies. Despite the remarkable impact that these therapies have had, there remains a significant proportion of people with cystic fibrosis (estimated at 10-15% of the global cystic fibrosis population) who are genetically ineligible for, or intolerant to, current CFTR-targeting therapies and whose therapeutic needs remain unmet. Inhaled genetic therapies offer the prospect of addressing the unmet pulmonary treatment need in people with cystic fibrosis, with several approaches, including gene addition therapy (the focus of this review), RNA-based therapies, antisense oligonucleotides and gene editing, being explored. Various non-viral and viral vectors have been investigated for cystic fibrosis gene addition therapy for mutation-agnostic restoration of CFTR function in the lungs. Lentiviral vectors offer the prospect of highly efficient and long-lasting gene expression, and the potential to be safely and, in contrast to other commonly used viral vectors, effectively re-dosed. A third-generation lentiviral vector pseudotyped with Sendai virus F and HN envelope proteins (rSIV.F/HN) has been developed for the treatment of cystic fibrosis. Promising preclinical results support the progression of this vector carrying a full-length CFTR transgene (BI 3720931) into a first-in-human clinical trial expected to begin in 2024.

Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats are challenging for several reasons, such as difficulty in locating and capturing them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation, but cells in frozen tissue are usually damaged and have low integrity and viability. Isolating primary cells from frozen tissues thus poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this is the first protocol specifically focused on fibroblast isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Primary fibroblast culture and subculture Support Protocol 3: Determination of growth curve and doubling time Support Protocol 4: Cell banking and thawing of primary fibroblasts Basic Protocol 3: Lentiviral transduction of bat primary fibroblasts Basic Protocol 4: Bat stable fibroblast cell line development Support Protocol 5: Bat fibroblast validation by immunofluorescence staining Basic Protocol 5: Chromosome counting.

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor-binding domain to direct lentivirus infection of Spike-expressing cells. Using four different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. Targeting is dependent on the fusion function of the Spike protein, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery by using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections.IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

Transduction of producer cells during lentiviral vector (LVV) production causes the loss of 70-90% of viable particles. This process is called retro-transduction and it is a consequence of the interaction between the LVV envelope protein, VSV-G, and the LDL receptor located on the producer cell membrane, allowing lentiviral vector transduction. Avoiding retro-transduction in LVV manufacturing is crucial to improve net production and, therefore, the efficiency of the production process. Here, we describe a method for quantifying the transduction of producer cells and three different strategies that, focused on the interaction between VSV-G and the LDLR, aim to reduce retro-transduction.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity in vitro and Venus fluorescence in vivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/β-catenin/TCF- and IL-1β/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and β-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.

The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for in vivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of \"ready-to-work\" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted in vivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications.

Gene therapy has made considerable strides in recent years. More than 4000 protein-coding genes have been implicated in more than 6000 genetic diseases; next-generation sequencing has dramatically revolutionized the diagnosis of genetic diseases. Most genetic diseases are considered very rare or ultrarare, defined here as having fewer than 1:100,000 cases, but only one of the 12 approved gene therapies (excluding RNA therapies) targets an ultrarare disease. This article explores three gene supplementation therapy approaches suitable for various rare genetic diseases: lentiviral vector-modified autologous CD34+ hematopoietic stem cell transplantation, systemic delivery of adeno-associated virus (AAV) vectors to the liver, and local AAV delivery to the cerebrospinal fluid and brain. Together with RNA therapies, we propose a potential business model for these gene therapies.

The purpose of this protocol is to provide a comprehensive, stepwise guide for assessing mitophagy flux utilizing a live-cell mt-KEIMA approach. The proposed protocol is sensitive, reproducible, quantitative, and easy to perform. While mitophagy has been extensively studied, current methodologies primarily focus on terminal measurements, neglecting the dynamic aspect of this process. Hence, the introduction of this straightforward live-cell mitophagy tracing protocol enables real-time monitoring of the dynamics of mitochondrial selective autophagy, thereby enhancing the ability to draw conclusions regarding key regulators and the reversibility of the process. The assay employs a lentiviral approach to induce mt-KEIMA expression in primary or immortalized cell lines. Subsequently, the respective mitophagy reporter cells are observed using a live-cell imaging system at specific time intervals, and further quantification allows the detection of mitophagy flux. This protocol has proven efficacious in investigating mitophagy flux, including responses to chemical inducers or genetically modified cells over time. Notably, this approach is well-suited for large throughput screening of chemicals or appropriate gene-editing libraries that may influence mitophagy responses in cells.

A 9-year-old boy with adrenoleukodystrophy due to ABCD1 whole-gene deletion was diagnosed with active cerebral adrenoleukodystrophy characterized by demyelination and gadolinium enhancement on brain MRI. He underwent hematopoietic cell transplant (HCT) with autologous CD34+ cells transduced with an ABCD1-expressing lentiviral vector (eli-cel [elivaldogene autotemcel]) as part of the ALD-104 clinical trial. Fifty days after HCT, the patient\'s MRI showed gadolinium resolution; the whole-blood vector copy number (VCN) was 0.666 copies/mL. Six months following HCT, an MRI showed re-emergence of gadolinium enhancement; the VCN had decreased to 0.029 copies/mL. Polyclonal antibodies to the ABCD1 gene product were detectable 9 months after transplant, showing reactivity to peroxisomes, suggesting an immune response; however, no antibody binding to human CD34+ cells could be shown. The patient underwent a successful allogeneic HCT 12 months after gene therapy with resultant gadolinium resolution, cerebral disease stabilization, and the disappearance of antibodies. The coincident VCN loss and appearance of antibody to the ABCD1 gene product is of interest, and we postulate that it is related to the patient\'s whole ABCD1 gene deletion. We suggest close monitoring of loss of gene therapy efficacy due to immune response in patients with full deletions who are considering gene therapy.