Respiratory diseases are a major global health issue, with a 38.9% increase in prevalence since 1990. Over 4 millions of patients die annually worldwide. Key diseases include asthma, COPD, acute lower respiratory tract infections, lung cancer, and tuberculosis, collectively affecting over 1 billion people. Lung transplantation, the only definitive treatment for end-stage lung diseases, faces challenges like low survival rates and organ shortages, with a five-year survival rate of 55% and high mortality rates while awaiting transplantation. One alternative solution can be cell therapy to treat the disease in situ and avoid the need of transplantation. This thesis aims at developing new strategies for lung repair and regeneration, focusing on targeted removal of dysfunctional epithelial cells and replacing them with therapeutic cells to restore alveolar homeostasis and halt disease progression. Two distinct approaches using animal models are investigated. 1. In Vivo De-Epithelialization in a Rat Model: Regional de-epithelialization was achieved using a mild detergent solution, selectively removing about 20% of the lower left lung lobe's epithelium without causing respiratory distress or significant mortality. This led to extensive epithelial cell proliferation and complete recovery of the alveolar epithelium by day 10, facilitated by endogenous lung progenitors. As proof of concept, the de-epithelialization model is used to study the engraftment of human therapeutical cells, exogenous distal lung epithelial progenitors (DLEPs). Patchy areas with engraftment of human alveolar epithelium were achieved in several rats and shown to significantly reduce lung injury scores in treated rats respect to control, indicating effective lung repair. 2. Targeted Removal and Replacement of Defective ATII Cells in Sftpc-/- Mouse Model: A novel fusion protein, DT388-SPA, was engineered by combining surfactant protein (SP) A, which is secreted and taken up by ATII cells, with the active domain of diphtheria toxin (DT388). DT388SPA was designed to selectively ablate defective ATII cells, enabling the introduction of healthy cells. As such, SPA acts as a “Trojan Horse” to deliver the toxin (DT388) into ATII cells, where it induces apoptosis. DT388-SPA demonstrated high specificity for ATII cells in vitro and in vivo, significantly reducing defective ATII cells and facilitating engraftment of healthy ATII cells, improving lung function and repair. Moreover, testing on human lung samples confirmed the efficacy and specificity of DT388-SPA, supporting its potential for clinical application. This thesis presents innovative approaches for lung disease treatment, focusing on in situ lung repair and regeneration. The de-epithelialization strategy and DT388-SPA technology show promise in reducing the need for lung transplants by directly repairing damaged lungs. Further research should optimize these methods and translate them into clinical practice to offer transformative therapies for patients with respiratory diseases.
Le malattie respiratorie sono una delle principali problematiche della salute a livello globale, con un aumento del 38,9% rispetto al 1990. Nel mondo si contano circa 4 milioni di pazienti che muoiono ogni anno. Asma, COPD, infezioni acute del tratto respiratorio inferiore, cancro ai polmoni e tubercolosi ne sono un esempio e collettivamente colpiscono oltre 1 miliardo di persone. Il trapianto di polmone è l'unico trattamento definitivo per le malattie polmonari allo stadio terminale ma è fortemente limitato dalla carenza di organi disponibili. Inoltre ha un tasso di sopravvivenza a cinque anni dal trapianto del solo 55% ed alti i tassi di mortalità durante l'attesa del trapianto. Una alternativa interessante al trapianto potrebbe essere la terapia cellulare per trattare la malattia in situ ed evitare la necessità del trapianto. Questa tesi mira a sviluppare nuove strategie per guarire e rigenerare l’epitelio polmonare, concentrandosi sulla rimozione mirata di specifiche cellule epiteliali disfunzionali e sulla loro sostituzione con cellule terapeutiche per ripristinare l'omeostasi alveolare e arrestare la progressione della malattia. La tesi è stata sviluppata studiando e sfruttando due approcci distinti su modelli animali. 1. De-epitelializzazione in vivo in ratto: La de-epitelializzazione è stata applicata iniettando un detergente per rimuovere selettivamente circa il 20% dell'epitelio del lobo inferiore sinistro del polmone senza causare distress respiratorio o mortalità nei soggetti testati. Questo ha stimolato una proliferazione delle cellule epiteliali delle zone adiacenti a quella trattata, per ripristinare completamente attraverso le cellule staminali endogene l'epitelio alveolare entro 10 giorni dal trattamento. La de-epitelializzazione è utilizzata anche per studiare il trapianto di cellule umane, i progenitori epiteliali distali polmonari (DLEP), confermandone l’efficacia nel trattamento di danni all’epitelio polmonare. 2. Rimozione mirata e sostituzione dei pneumociti di tipo 2 difettosi in topi Sftpc-/-: Una nuova proteina, DT388-SPA, è stata progettata combinando la proteina del surfattante (SP) A, fisiologicamente secreta e assorbita dai pneumociti di tipo 2, con il dominio attivo della tossina difterica (DT388). DT388-SPA è stata progettata per rimuovere selettivamente solo i pneumociti di tipo 2 difettosi, consentendo l'introduzione di cellule sane. SPA funge da "Cavallo di Troia" per consegnare la tossina (DT388) alle cellule, dove ne induce l'apoptosi. DT388-SPA ha dimostrato un'alta specificità per i pneumociti in vitro e in vivo, riducendone significativamente il numero e facilitando il trapianto di cellule sane, migliorando la funzione polmonare. Inoltre, i test su campioni di biopsie polmonari umane hanno confermato l'efficacia e la specificità di DT388-SPA, supportando il suo potenziale per l'applicazione clinica. Questa tesi riporta approcci innovativi per il trattamento delle malattie polmonari, concentrandosi sulla guarigione e rigenerazione dell’epitelio polmonare in situ. La strategia di de-epitelializzazione e l’uso della proteina DT388-SPA possono essere promettenti per ridurre la necessità di trapianti trattando direttamente i polmoni danneggiati con trapianti cellulati. Ulteriori test ed analisi per ottimizzare questi metodi sono necessari per poterli poi tradurre nella pratica clinica.
Targeted approaches to promote repair and regeneration in epithelial lung disease
Predella, Camilla
2023/2024
Abstract
Respiratory diseases are a major global health issue, with a 38.9% increase in prevalence since 1990. Over 4 millions of patients die annually worldwide. Key diseases include asthma, COPD, acute lower respiratory tract infections, lung cancer, and tuberculosis, collectively affecting over 1 billion people. Lung transplantation, the only definitive treatment for end-stage lung diseases, faces challenges like low survival rates and organ shortages, with a five-year survival rate of 55% and high mortality rates while awaiting transplantation. One alternative solution can be cell therapy to treat the disease in situ and avoid the need of transplantation. This thesis aims at developing new strategies for lung repair and regeneration, focusing on targeted removal of dysfunctional epithelial cells and replacing them with therapeutic cells to restore alveolar homeostasis and halt disease progression. Two distinct approaches using animal models are investigated. 1. In Vivo De-Epithelialization in a Rat Model: Regional de-epithelialization was achieved using a mild detergent solution, selectively removing about 20% of the lower left lung lobe's epithelium without causing respiratory distress or significant mortality. This led to extensive epithelial cell proliferation and complete recovery of the alveolar epithelium by day 10, facilitated by endogenous lung progenitors. As proof of concept, the de-epithelialization model is used to study the engraftment of human therapeutical cells, exogenous distal lung epithelial progenitors (DLEPs). Patchy areas with engraftment of human alveolar epithelium were achieved in several rats and shown to significantly reduce lung injury scores in treated rats respect to control, indicating effective lung repair. 2. Targeted Removal and Replacement of Defective ATII Cells in Sftpc-/- Mouse Model: A novel fusion protein, DT388-SPA, was engineered by combining surfactant protein (SP) A, which is secreted and taken up by ATII cells, with the active domain of diphtheria toxin (DT388). DT388SPA was designed to selectively ablate defective ATII cells, enabling the introduction of healthy cells. As such, SPA acts as a “Trojan Horse” to deliver the toxin (DT388) into ATII cells, where it induces apoptosis. DT388-SPA demonstrated high specificity for ATII cells in vitro and in vivo, significantly reducing defective ATII cells and facilitating engraftment of healthy ATII cells, improving lung function and repair. Moreover, testing on human lung samples confirmed the efficacy and specificity of DT388-SPA, supporting its potential for clinical application. This thesis presents innovative approaches for lung disease treatment, focusing on in situ lung repair and regeneration. The de-epithelialization strategy and DT388-SPA technology show promise in reducing the need for lung transplants by directly repairing damaged lungs. Further research should optimize these methods and translate them into clinical practice to offer transformative therapies for patients with respiratory diseases.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/221894