Modulation of lung development by In utero gene transfer

Advances in prenatal diagnosis of genetic and congenital disorders with progressively more sensitive techniques may increase opportunities for consideration of prenatal gene therapy. There are a number of genetic and acquired disorders with peri or postnatal pulmonary manifestations. These include monogenetic diseases like cystic fibrosis or surfactant protein B deficiency that would presumably require long-term expression of the deficient or defective gene. However, there are also abnormalities of lung growth, such as congenital diaphragmatic hernia, or lung maturation, such as respiratory distress syndrome of prematurity, that could potentially benefit from strategies that achieve transient gene expression in specific pulmonary distributions. Considered an attractive target organ for fetal gene transfer, the developing lung, poses also some obstacles that would only be overcomed with the development of a variety of gene transfer methodologies: different types of vector, optimal site, route and timing of gene delivery. The fundament of this dissertation was to modulate lung growth/maturation by in utero gene transfer, aiming to unveil underlying mechanisms of normal and abnormal lung development. The first objective of this dissertation was to develop a new approach of gene transfer targeting the fetal lung in early stages of lung development. We developed a new method for direct injection of viral vectors into the rat fetal lung as early as the pseudoglandular phase of lung development using ultrasound guided microinjections. The pseudoglandular stage, characterized by intense branching morphogenesis, is the period of greatest overall growth of the airways and vasculature of the fetal lung and corresponds to a stage of immunologic immaturity. Therefore, gene transfer during this period has the potential to have major effects on key elements of lung growth with minimal potential for detrimental immune responses. We aimed to compare two distinct types of vectors: an adenoviral vector and a lentiviral vector (equine infectious anemia virus-based), both expressing the enhanced green fluorescent protein reporter gene. This study confirmed that adenoviral vectors are more suitable when rapid, high-level and transient expression of the transgene is required; whereas lentiviral vectors are more appropriate to induce sustained and long-term expression. One of the concerns in gene transfer protocols is to target a specific cellular compartment of a determined organ/system. Interestingly, interstitial compartment rather than epithelial cells were transduced in opposition to previous studies describing intrapulmonary, intraamniotic and intratracheal administrations of viral vectors. The observation of transduction of distinct cell populations within the lung with different routes of transduction raises the possibility of manipulating gene expression in specific and separate cell populations within the developing lung. We then decided to use this model system to perform an in vivo study of dynamic lung morphogenesis, involving a major player in branching morphogenesis, fibroblast growth factor 10 (FGF10). We observed that FGF10 mesenchymal overexpression, on the fetal rat lung, resulted in the induction of consistent patterns of malformation, the appearance of which were developmental stage and location dependent. These malformations, in total, appear to closely recapitulate the morphology and histology of the entire spectrum of human Congenital Cystic Adenomatoid Malformation (CCAM).

Name thesis:degree_name

Tese de doutoramento em Ciências da Saúde (ramo de conhecimento em Ciências Biológicas e Biomédicas)

Year dc:date.issued

2009

Author dc:creator

• Santos, Sílvia Gonzaga da Silva

Advisors dc:contributor.advisor

• Correia-Pinto, Jorge
• Flake, Alan Wayne

Statement dc:rights

• openAccess

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1822/9866

OAI record oai:identifier

http://repositorium.sdum.uminho.pt/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arepositorium.uminho.pt%3A1822%2F9866