Integrating Viral and Nonviral Vectors for Cystic Fibrosis Gene Therapy in the Airways

An important goal for cystic fibrosis (CF) gene therapy is to achieve long-term functional correction. While many vector options have been evaluated, integrating vectors have the greatest potential to maintain stable expression over time without a requirement for repeated administration. In this chapter, we discuss the importance of correcting the appropriate cell types, options for integrating vectors, animal models for CF gene therapy, and clinically relevant endpoint measurements. Lentiviral vectors are a promising option for CF gene therapy, as they integrate into the host genome and persistently express a transgene of interest. Airway cell tropism can be conferred by pseudotyping. Nonviral vectors such as DNA transposons can also integrate into the genome. Recent advances in hybrid viral/transposon vector technology improve the ability to deliver transposons to the airways in vivo. Integrating vector technology and new animal models have allowed considerable progress toward the goal of using gene therapy to correct life-long genetic diseases such as CF

Gene therapy potential for genetic disorders of surfactant dysfunction

Pulmonary surfactant is critically important to prevent atelectasis by lowering the surface tension of the alveolar lining liquid. While respiratory distress syndrome (RDS) is common in premature infants, severe RDS in term and late preterm infants suggests an underlying genetic etiology. Pathogenic variants in the genes encoding key components of pulmonary surfactant including surfactant protein B (SP-B

Monocytic/Macrophagic Pneumonitis after Intrabronchial Deposition of Vascular Endothelial Growth Factor in Neonatal Lambs

Preterm and young neonates are prone to inadequate surfactant production and are susceptible to respiratory distress syndrome characterized by alveolar damage and hyaline-membrane formation. Glucocorticoid therapy is commonly used in preterm and young infants to enhance lung maturation and surfactant synthesis. Recently, vascular endothelial growth factor (VEGF) was suggested to be a novel therapeutic agent for lung maturation that lacked adverse effects in mice. The purpose of this study was to assess the safety of incremental concentration (0.0005, 0.005, and 0.05 mg/ml) and duration (16, 24, and 32 hours) of recombinant human VEGF after bronchoscopic instillation (10 ml) in neonatal lambs. High-dose VEGF caused locally extensive plum-red consolidation that was microscopically characterized by interstitial and alveolar infiltrates of cells that were morphologically and phenotypically (CD68+) consistent with monocytes/macrophages. T cells (CD3+) and B cells (CD79+) were located primarily in bronchus/bronchiole-associated lymphoid tissue and were not consistently altered by treatment with VEGF. The dose of VEGF had significant effects on both gross lesions (P \u3c .0047) and microscopic monocyte/macrophage recruitment scores (P \u3c .0001). Thus, the VEGF dose instilled into the lung greatly influenced cellular recruitment and lesion development. The post-dosing interval of VEGF in this study had minor impact (no statistical significance) on cellular recruitment. This study showed that airway deposition of VEGF in the neonatal lamb induces monocyte/macrophage recruitment to the lung and high doses can cause severe lesions. The cellular recruitment suggests further research is needed to define dosages that are efficacious in enhancing lung maturation while minimizing potential adverse effects

PLUNC Is a Novel Airway Surfactant Protein with Anti-Biofilm Activity

The PLUNC ("Palate, lung, nasal epithelium clone") protein is an abundant secretory product of epithelia present throughout the conducting airways of humans and other mammals, which is evolutionarily related to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family. Two members of this family--the bactericidal/permeability increasing protein (BPI) and the lipopolysaccharide binding protein (LBP)--are innate immune molecules with recognized roles in sensing and responding to Gram negative bacteria, leading many to propose that PLUNC may play a host defense role in the human airways.Based on its marked hydrophobicity, we hypothesized that PLUNC may be an airway surfactant. We found that purified recombinant human PLUNC greatly enhanced the ability of aqueous solutions to spread on a hydrophobic surface. Furthermore, we discovered that PLUNC significantly reduced surface tension at the air-liquid interface in aqueous solutions, indicating novel and biologically relevant surfactant properties. Of note, surface tensions achieved by adding PLUNC to solutions are very similar to measurements of the surface tension in tracheobronchial secretions from humans and animal models. Because surfactants of microbial origin can disperse matrix-encased bacterial clusters known as biofilms [1], we hypothesized that PLUNC may also have anti-biofilm activity. We found that, at a physiologically relevant concentration, PLUNC inhibited biofilm formation by the airway pathogen Pseudomonas aeruginosa in an in vitro model.Our data suggest that the PLUNC protein contributes to the surfactant properties of airway secretions, and that this activity may interfere with biofilm formation by an airway pathogen

Supernova hydrodynamics experiments on the Nova laser

In studying complex astrophysical phenomena such as supernovae, one does not have the luxury of setting up clean, well-controlled experiments in the universe to test the physics of current models and theories. Consequently, creating a surrogate environment to serve as an experimental astrophysics testbed would be highly beneficial. The existence of highly sophisticated, modern research lasers, developed largely as a result of the world-wide effort in inertial confinement fusion, opens a new potential for creating just such an experimental testbed utilizing well-controlled, well-diagnosed laser-produced plasmas. Two areas of physics critical to an understanding of supernovae are discussed that are amenable to supporting research on large lasers: (1) compressible nonlinear hydrodynamic mixing and (2) radiative shock hydrodynamics. © 1997 American Institute of Physics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69962/2/PHPAEN-4-5-1994-1.pd

Laser experiments to simulate supernova remnants

An experiment using a large laser facility to simulate young supernova remnants (SNRs) is discussed. By analogy to the SNR, the laboratory system includes dense matter that explodes, expansion and cooling to produce energetic, flowing plasma, and the production of shock waves in lower-density surrounding matter. The scaling to SNRs in general and to SN1987A in particular is reviewed. The methods and results of x-ray radiography, by which the system in diagnosed, are discussed. The data show that the hohlraum used to provide the energy for explosion does so in two ways—first, through its radiation pulse, and second, through an additional impulse that is attributed to stagnation pressure. Attempts to model these dynamics are discussed. © 2000 American Institute of Physics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69889/2/PHPAEN-7-5-2142-1.pd

Ferret and Pig Models of Cystic Fibrosis: Prospects and Promise for Gene Therapy

Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies