01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

(Prehn et al, 1996); this implies a potential avenue of
TGF-β1 gene transfer for Alzheimer's disease.
Transfer of TGF-β1 cDNA in vivo suppresses local T
cell immunity and prolonged cardiac allograft survival in
mice; TGF-β1 gene transfer may become a new type of
immunosuppressant avoiding the systemic toxicity of
conventional immunosuppression (Qin et al, 1996).
GM-CSF overexpression induced TGF-β1 gene
expression and secretion from macrophages purified from
bronchoalveolar lavage fluid 7 days after GM-CSF gene
transfer; these findings implicate GM-CSF in pulmonary
fibrogenesis (Xing et al, 1997).
XXXIII. Cystic fibrosis (CF)
A. Molecular mechanism of CF
pathogenesis
CF is a lethal recessive hereditary disorder
characterized by abnormalities of the airway epithelium; it
affects 1 in 2,000 Caucasians. Inflicted individuals show
secretion of thick mucus and chronic colonization of the
lung epithelium with pathogens such as Pseudomonas
aeruginosa. The defect arises from mutations in the 250kb
gene encoding a 12-transmembrane domain
glycoprotein (1480 amino acids), called cystic fibrosis
transmembrane conductance regulator (CFTR), that
modulates the permeability of Cl - in response to elevation
of intracellular cAMP. The defect is caused by deletion of
three base pairs eliminating a single phenylalanine residue
at the center of the first nucleotide-binding domain of the
CFTR protein (Riordan et al, 1989).
Much of the mortality seen in CF is related to chronic
infection of the respiratory tract with Pseudomonas
aeruginosa; Pseudomonas colonization has been
attributed to increased numbers of specific cell-surface
receptors and to the presence of mucus. Adherence of P.
aeruginosa directly to the cell surface of CF airway
epithelium (from noncultured nasal epithelial cells isolated
from CF patients) was significantly increased over that in
cells from healthy donors. Liposome-mediated CFTR gene
transfer resulted in a significant reduction in the numbers
of bacteria bound to ciliated CF epithelial cells (Davies et
al, 1997).
The architecture of the lung and the terminal
differentiation of the defective cells imposes a serious
hurdle for ex vivo gene therapy for CF: the epithelial cells
on the airway surface cover the successively branching
structures of the lung making impossible their removal and
reimplantation (Yoshimura et al, 1992).
Successful introduction of the entire 250 kb human
CFTR gene locus and adjacent sequences into Chinese
hamster ovary-K1 (CHO) cells which lack endogenous
CFTR was achieved using yeast artificial chromosomes
(YACs); integration of the human CFTR-containing YACs
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into the CHO genome took place on the order of one copy
per genome; functional human CFTR was expressed from
subclones and human CFTR expression in CHO cells was
unexpectedly high (Mogayzel et al, 1997). This type of
studies are very useful as a number of DNA control
elements for CFTR may be “hidden” throughout the gene
locus, including enhancers, ORIs, silencers, MARs, that
participate in the tissue- and developmental stage-specific
CFTR gene expression.
The airway epithelium is in the process of injury and
regeneration in CF; regenerating poorly differentiated cells
of human airway epithelium in culture were efficiently
transfected with CFTR cDNA using adenoviral vectors;
CFTR expression and cAMP-regulated stimulation of the
cell membrane chloride ion secretion took place on these
cells as determined by light fluorescence microscopy and
scanning laser confocal microscopy (Dupuit et al, 1997).
B. CFTR gene transfer in animal models
Direct transfer of the human CFTR gene was achieved
using a replication-defective adenovirus vector by
intratracheal instillation into cotton rat lungs; the presence
of human CFTR mRNA transcripts was detected by in situ
hybridization with a cRNA (antisense) probe as well as by
immunohistochemical evaluation using antibodies directed
against the CFTR protein (Rosenfeld et al, 1992).
First generation adenovirus-mediated gene transfer of
CFTR to the mouse lung resulted in the expression of viral
proteins leading to the elimination of the therapeutic cells
expressing CFTR by cellular immune responses; second
generation E1-deleted viruses displayed substantially
longer recombinant gene expression and induced a lower
inflammatory response (Yang et al, 1994).
Adenoviral vector constructs with an E1-
E3+E4ORF6+ backbone encoding CFTR (or βgalactosidase)
produced declining levels of expression
while a similar vector with an E1-E3+E4+ backbone gave
rise to sustained, long-term reporter gene expression in the
lung in nude mice; CTLs directed against either adenoviral
proteins or β-galactosidase reduced expression in nude
mice stably expressing β-galactosidase from the E4+
vector (Kaplan et al, 1997).
Aerosol delivery of an adenoviral vector encoding
CFTR to non-human primates showed human CFTR
mRNA in lung tissue from all treated animals on days 3, 7,
and 21 post-exposure; other than some rather mild
complications on individual animals ranging from an
increase in lavage lymphocyte numbers to
bronchointerstitial pneumonia, the treatment was rather
safe (McDonald et al, 1997).
Adenoviruses elicit am immune response. Effective
gene therapy for CF would ideally be accomplished with a
vector capable of long-term expression of the CFTR in the
absence of a host inflammatory response; in this respect