The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc + Int − ) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc + Int − transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int − phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc + Int − transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.NA "cut-and-paste" transposable elements are important tools for genome engineering, such as insertional mutagenesis and transgenesis. Research with the DNA transposon Sleeping Beauty, a "resurrected" transposon, has pioneered the use of DNA transposons in mammalian cells (1, 2). piggyBac is also a DNA transposon and a promising alternative to Sleeping Beauty. piggyBac, originally isolated from the cabbage looper moth Trichoplusia ni genome (3), has a large cargo size (4), is highly active in many cell types, and mediates long-term expression in mammalian cells in vivo (5-10). piggyBac is also distinguished by its ability to excise precisely (11), thus restoring the donor site to its pretransposon insertion sequence.Because it can excise precisely, piggyBac is especially useful if a transgene is only transiently required. Transient integration and expression of transcription factors are important approaches to generate transgene-free induced pluripotent stem cells (iPSCs) (12, 13) as well as directed differentiation of specific cell types for both research and clinical use. Removal of the transgenes is key for potential therapeutic applications of iPSCs. piggyBac has been used as a vector for reversible integration; however, reintegration of the transposon catalyzed by piggyBac (PB) transposase occurs in 40-50% of cells (14) Int− transposase whose excision frequency is five-to six-f...

Lassa virus (LASV) is an Old World arenavirus responsible for hundreds of thousands of infections in West Africa every year. LASV entry into a variety of cell types is mediated by interactions with glycosyltransferase LARGE-modified O-linked glycans present on the ubiquitous receptor α-dystroglycan (αDG). However, cells lacking αDG are permissive to LASV infection, suggesting that alternative receptors exist. Previous studies demonstrated that the phosphatidylserine (PtdSer)-binding receptors Axl and Tyro3 along with C-type lectin receptors mediate αDG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP)-pseudotyped virion entry into αDG-knocked-out HEK 293T and wild-type (WT) Vero cells, which express αDG lacking appropriate glycosylation. To investigate the mechanism by which TIM-1 mediates enhancement of entry, we demonstrate that mutagenesis of the TIM-1 IgV domain PtdSer-binding pocket abrogated transduction. Furthermore, the human TIM-1 IgV domain-binding monoclonal antibody ARD5 blocked transduction of pseudovirions bearing LASV GP in a dose-dependent manner. Finally, as we showed previously for other viruses that use TIM-1 for entry, a chimeric TIM-1 protein that substitutes the proline-rich region (PRR) from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional αDG, TIM-1 mediates the entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer-binding pocket of TIM-1. PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through the binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate the entry of all enveloped viruses, yet LASV GP-pseudotyped viruses were previously found to be unresponsive to PtdSer receptor enhancement in HEK 293T cells. Here, we demonstrate that LASV pseudovirions can utilize the PtdSer receptor TIM-1 but only in the absence of appropriately glycosylated α-dystroglycan (αDG), the high-affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why previous studies performed with α-DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cAMP-regulated anion channel. Although CF is a multi-organ system disease, most people with CF die of progressive lung disease that begins early in childhood and is characterized by chronic bacterial infection and inflammation. Nearly 90% of people with CF have at least one copy of the ΔF508 mutation, but there are hundreds of CFTR mutations that result in a range of disease severities. A CFTR gene replacement approach would be efficacious regardless of the disease-causing mutation. After the discovery of the CFTR gene in 1989, the in vitro proof-of-concept for gene therapy for CF was quickly established in 1990. In 1993, the first of many gene therapy clinical trials attempted to rescue the CF defect in airway epithelia. Despite the initial enthusiasm, there is still no FDA-approved gene therapy for CF. Here we discuss the history of CF gene therapy, from the discovery of the CFTR gene to current state-of-the-art gene delivery vector designs. While implementation of CF gene therapy has proven more challenging than initially envisioned; thanks to continued innovation, it may yet become a reality.

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in CF transmembrane conductance regulator (CFTR), resulting in defective anion transport. Regardless of the disease-causing mutation, gene therapy is a strategy to restore anion transport to airway epithelia. Indeed, viral vector–delivered CFTR can complement the anion channel defect. In this proof-of-principle study, functional in vivo CFTR channel activity was restored in the airways of CF pigs using a feline immunodeficiency virus–based (FIV-based) lentiviral vector pseudotyped with the GP64 envelope. Three newborn CF pigs received aerosolized FIV-CFTR to the nose and lung. Two weeks after viral vector delivery, epithelial tissues were analyzed for functional correction. In freshly excised tracheal and bronchus tissues and cultured ethmoid sinus cells, we observed a significant increase in transepithelial cAMP-stimulated current, evidence of functional CFTR. In addition, we observed increases in tracheal airway surface liquid pH and bacterial killing in CFTR vector–treated animals. Together, these data provide the first evidence to our knowledge that lentiviral delivery of CFTR can partially correct the anion channel defect in a large-animal CF model and validate a translational strategy to treat or prevent CF lung disease.

The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR.