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).
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.
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
b) to apply and exploit new viral and non-viral gene transfer vectors
to the CF lung disease.
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
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.
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
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.
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.
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;
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.
M, Assael BM Bacterial infections and inflammation in the lungs
of cystic fibrosis patients. Pediatr Infect Dis J, 20: 207-213,
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.
M, Conese M. Non-viral approach toward gene therapy of cystic fibrosis
lung disease. Curr Gene Ther, 2002 in press..
C, Carpani D, Conese M, Caci E, Zegarra de Moran O, Donini P Ascenzioni
Functional human CFTR produced by a stable minichromosome. Submitted