Gene Delivery to the Airway

Keiser, Nicholas W.; Engelhardt, John F.

doi:10.1002/0471142905.hg1309s78

Cited by 8 publications

(5 citation statements)

References 76 publications

(141 reference statements)

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“…To test the downstream application of 3T31Y-expanded airway epithelium in vivo, we used a xenogeneic engraftment model (46,47) to repopulate denuded tracheal grafts ( Figure E12). We processed rat tracheal scaffolds using repeated freeze-thaw cycles (48) and generated a matrix depleted of endogenous epithelium ( Figure 7A, left).…”

Section: Repopulation Of Tracheal Scaffolds In a Xenograft Modelmentioning

confidence: 99%

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

Butler

Hynds

Gowers

et al. 2016

Am J Respir Crit Care Med

181

210

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Rationale: Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts.Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts.Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T31Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures.

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Section: Repopulation Of Tracheal Scaffolds In a Xenograft Modelmentioning

confidence: 99%

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

Butler

Hynds

Gowers

et al. 2016

Am J Respir Crit Care Med

181

210

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“…28 Tenfold higher transduction was also observed using rAAV2/HBoV1 in HAE-ALI cultures derived from primary airway cells versus in the immortalized human airway cell line CuFi8. 36 Reasoning that the extent of differentiation of FAE-ALI cultures may affect the findings, the study was extended to an ex vivo tracheal xenograft model in which subcutaneous implantation of human 44,47 and ferret 46 proximal airway epithelia into athymic mice produces better differentiated epithelia than the ALI cultures. Both the human and ferret xenografts were infected with equal amounts of a mixture of two rAAV2/HBoV1 vectors, one carrying the secreted Gaussia luciferase (gLuc) and the other carrying intracellular fLuc.…”

Section: Results Raav2/hbov1 Transduction Of Ferret Versus Human Airway Epithelium Using In Vitro and Ex Vivo Modelsmentioning

confidence: 99%

“…Human and ferret tracheal xenografts were generated as subcutaneous implants in athymic mice, and were used as an ex vivo model of a vascularized airway for virus infection. As previously described, 44 human tracheal xenografts were generated by seeding the denuded rat tracheas with primary human bronchial airway cells; ferret tracheal xenografts were generated directly from around a 1 cm segment of 2-day-old ferret trachea. 45,46 The denuded rat tracheas or segments of fresh ferret trachea were cannulated with flexible plastic tubing, and the assembled tracheal cassettes were transplanted subcutaneously into athymic mice.…”

Section: Infection Of Xenograftsmentioning

confidence: 99%

Human Bocavirus Type-1 Capsid Facilitates the Transduction of Ferret Airways by Adeno-Associated Virus Genomes

et al. 2017

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Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such, this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However, preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets, predominantly in the distal airways. Analyses of in vivo, ex vivo, and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age, reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models.

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“…Future scRNA-seq studies to characterize other cellular changes in the cryopreservation recovery process should be informative for understanding mechanisms of airway regeneration. In vitro , airway basal cells can be efficiently transduced by gene transfer vectors for genetic modification 34,35 . Thus, unobstructed access to the basal cell layer following cryo-injury could facilitate gene modulation in ex vivo human tissues, a unique application of this technology.…”

Section: Discussionmentioning

confidence: 99%

Prolonged airway explant culture enables study of health, disease, and viral pathogenesis

Lee-Ferris,

Okuda,

Galiger

et al. 2024

Preprint

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In vitro models play a major role in studying airway physiology and disease. However, the native lung’s complex tissue architecture and non-epithelial cell lineages are not preserved in these models. Ex vivo tissue models could overcome in vitro limitations, but methods for long-term maintenance of ex vivo tissue has not been established. We describe methods to culture human large airway explants, small airway explants, and precision-cut lung slices for at least 14 days. Human airway explants recapitulate genotype-specific electrophysiology, characteristic epithelial, endothelial, stromal and immune cell populations, and model viral infection after 14 days in culture. These methods also maintain mouse, rabbit, and pig tracheal explants. Notably, intact airway tissue can be cryopreserved, thawed, and used to generate explants with recovery of function 14 days post-thaw. These studies highlight the broad applications of airway tissue explants and their use as translational intermediates between in vitro and in vivo studies.

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Gene Delivery to the Airway

Cited by 8 publications

References 76 publications

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

Human Bocavirus Type-1 Capsid Facilitates the Transduction of Ferret Airways by Adeno-Associated Virus Genomes

Prolonged airway explant culture enables study of health, disease, and viral pathogenesis

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