Cystic Fibrosis Association

The Cystic Fibrosis Association (CFA, Associazione Lombarda Fibrosi Cistica) was initiated 25 years ago with the aim of actively supporting the Regional Centre for Cystic Fibrosis located within the De Marchi Hospital's Pediatrics Department in Milan, in assisting patients affected by cystic fibrosis.
In 1998 it obtained the status of a non-profit organisation ONLUS (Organizzazione Non Lucrativa di Utilità Sociale).

Since 2000, the Cystic Fibrosis Association has opened a new research facility (Institute for Experimental Treatment of Cystic Fibrosis) at the San Raffaele Science Park with the purpose of promoting scientific research in the field of gene therapy treatment of cystic fibrosis.
Cystic fibrosis (CF) is the principal fatal genetic disease in Caucasians (frequency disease: 1:2500 live births). CF affects a number of epithelial-lined organs, the most important being the lungs. Bacterial colonization of the bronchial tree occurs in childhood and progresses to episodes of overt infection which triggers an exaggerated inflammatory response and subsequent lung damage.

Since Cystic Fibrosis (CF) is an autosomal recessive disorder due to mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, studies towards a gene therapy approach to its treatment followed immediately upon the cloning of the gene. Encouraging results were obtained in many in vivo model systems (CF transgenic mice) involving viral as well as non-viral vectors, which demonstrated the recovery of CFTR function in the airways.
The Institute for Experimental Treatment of Cystic Fibrosis has several aims oriented:
a) to understand the barriers impeding gene delivery to respiratory epithelial cells;
b) to apply and exploit new viral and non-viral gene transfer vectors to the CF lung disease.

Clinical trials in CF patients carried out with cationic lipids have outlined that low transfection efficiency and poor maintenance of gene expression are so far the main obstacles to an effective CF gene therapy approach. We have then chosen to investigate the mechanism of gene delivery by alternative non-viral vectors, i.e. cationic polymers, among which polyethylenimine (PEI) is the most promising gene transfer vector.
A direct comparison between cationic lipids and polymers demonstrated the capacity of PEI to overcome the barriers imposed by the airway surface fluid. Studies on biodistribution of complexes after intratracheal injection revealed that lipoplexes (e.g. DOTAP/DNA) aggregated in the lumen of mouse bronchi and co-localized either totally or partially with the surfactant protein A (Figure 1A). Consistent with this, no GFP expression was found in the airway epithelial cells in lipoplexes-treated animals (Fig. 1B). On the contrary, polyplexes (PEI 25K/DNA) were only found within the bronchial cells (Fig. 1C) where transgene expression was also revealed (Fig. 1D). We have established in our laboratory a mouse model of acute respiratory infection with Pseudomona aseruginosa, a bacterium present in almost the totality of CF patients. We will assay the efficiency of PEI gene transfer in this model, which resembles the pathological conditions of CF lung disease.
CF inflamed airways are characterized by squamous metaplasia of the respiratory epithelium. Primay squamous nasal and bronchial cells show both decreased binding of cationic lipid DOTAP/DNA complexes and transgene levels respect to less differentiated cells, indicating that the interaction between the transfecting complexes and the plasma membrane is essential for the cationic carrier-mediated gene delivery to the CF airways.
These findings could in part explain why cationic liposomes have given a partial recovery of CFTR function in the human lungs and prompt to evaluate the efficiency and efficacy of PEI in human studies.

Among the viral vectors, lentiviruses address the problem of poor persistence due to their ability to integrate, and offer the advantage of infecting nondividing cells, a significant consideration in the airways where most cells are mitotically inactive.
Preliminary studies in fetal human airway xenografts have showed that last-generation lentiviral vectors could transduce 2-20% cells of a well-differentiated respiratory epithelium. Transgene expression was still present at comparable levels and with similar pattern at 12-16 weeks after lentivirus infection. The increased production of pro-inflammatory cytokines and a decreased bacterial internalization have been linked to the pathogenesis of CF infectious lung disease. The efficacy of CFTR-lentiviral vectors will be assayed in CF xenografts evaluating the production of cytokines and the recovery of bacterial internalisation.

The gene transfer vectors employed so far do not contain all the necessary regulatory elements to determine a CFTR expression which is correct in time and space. Artificial chromosomes offer some important advantages for CF gene therapy: they can host large fragments of exogenous DNA thus allowing the insertion of entire genetic loci; moreover, they are episomal and replicate autonomously, are stable retained in the host cells at one-two copies per cells, and are not immunogenic. A first version of an artificial chromosome derived from human chromosome 1 containing the CFTR coding and 5' upstream sequences, named CFTR-MC1, has already been assembled. We have demonstrated that the CFTR protein is expressed and it is functional in CHO cells. To show that this artificial chromosome is active in a gene therapy context, CFTR-MC1 will be introduced in CF respiratory epithelial cells in in vitro and in vivo models. Besides the study of CFTR protein localisation, particular emphasis will be given to the recovery of the pro-inflammatory and anti-bacterial activities of the airway epithelium.


Biffi A, Sersale G, Cassetti A, Villa A, Bordignon C, Assael BM, Conese M. Restoration of bacterial killing activity of human respiratory cystic fibrosis cells through cationic vector-mediated cystic fibrosis transmembrane conductance regulator gene transfer. Hum Gene Ther 1999; 10: 1923-1930.

Bragonzi A, Boletta , Biffi A, Muggia A, Sersale G, Cheng SH, Bordignon C, Assael BM, Conese M. Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs. Gene Ther 1999; 6: 1995-2004.

Bragonzi A, Dina G, Villa A, Calori G, Biffi A, Bordignon C, Assael BM, Conese M. Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs. Gene Ther 2000; 7: 1753-1760.

Conese M, Assael BM Bacterial infections and inflammation in the lungs of cystic fibrosis patients. Pediatr Infect Dis J, 20: 207-213, 2001.

Sersale G, Carpani D, Casotti V, Livraghi A, Carrabino S, Di Cicco M, Assael BM, Giunta A, Conese M. Inhibition of nonviral cationic liposome-mediated gene transfer into primary human respiratory cells by interferon-gamma. J Mol Med, 2002 in press.

Bragonzi M, Conese M. Non-viral approach toward gene therapy of cystic fibrosis lung disease. Curr Gene Ther, 2002 in press..

Auriche C, Carpani D, Conese M, Caci E, Zegarra de Moran O, Donini P Ascenzioni F
Functional human CFTR produced by a stable minichromosome. Submitted Revised.

Biological barriers to cationic vector-mediated gene transfer into the lungs