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

Figure 33. Immunofluorescent detection of in vitro fusion of
α-glucosidase (GAA)-deficient muscle cells with myoblasts
transduced with the GAA gene using a retroviral vector. The
GAA-transduced myoblasts (red fluorescence) were able to fuse
(arrows) with deficient myoblasts (green fluorescence) and
provide enzyme activity. From Zaretsky JZ, Candotti F,
Boerkoel C, Adams EM, Yewdell JW, Blaese RM, Plotz PH
(1997) Retroviral transfer of acid α-glucosidase cDNA to
enzyme-deficient myoblasts results in phenotypic spread of the
genotypic correction by both secretion and fusion. Hum Gene
Ther 8, 1555-1563. Reproduced with the kind permission of the
authors and Mary Ann Liebert, Inc.
further took place by cell-to-cell interaction between
transduced and nontransduced cells in culture suggesting
that only partial cell transduction might be sufficient to
correct AGA deficiency in vivo (Enomaa et al, 1995).
Mucopolysaccharidosis type VII (Sly syndrome or
MPS VII) is a result of an inherited deficiency of βglucuronidase
in humans, mice, and dogs (see Wolfe et al,
1992 for references). The symptoms are progressive
degeneration in several tissues resulting from storage in
lysosomes of undegraded glycosaminoglycans affecting
the spleen, liver, brain, cornea, kidney, and skeletal
muscle. Affected individuals display a reduced life-span
which is 5 months in mice. Retroviral vectors have
successfully treated mucopolysaccharidosis VII by
somatic cell gene transfer in mouse models (Wolfe et al,
1992) or by implantation of ex vivo modified mouse skin
fibroblasts (Moullier et al, 1993).
Retrovirus-mediated transfer of the iduronate-2sulfatase
cDNA into lymphocytes is being applied for the
clinical treatment of mild Hunter syndrome (mucopolysaccharidosis
type II, protocol # 65 on page 163).
XXXVII. Hereditary α1-antitrypsin
(α1-AT) deficiency
Destruction of components of the extracellular matrix
of the lung by neutrophil elastase (NE) is believed to be a
critical event in the development of obstructive lung
disease. α1-antitrypsin (also known as α1-proteinase
inhibitor) is an antiprotease that protects the lung from
destruction by the powerful protease NE. The hereditary
α1-antitrypsin deficiency is caused by mutations in the
coding region of the 12.2 kb-gene resulting in decreased
serum and lung levels of α1-antitrypsin; affected
individuals develop emphysema at age 30-40 (Crystal,
1990). Lung-derived epithelial cells have the capacity to
synthesize functional α1-antitrypsin but also to increase
the rate of its production when stimulated by specific
inflammatory mediators, including oncostatin M, IL-1,
and dexamethasone (Cichy et al, 1997).
The respiratory epithelium has been a potential site for
somatic gene therapy because of the possibility of direct
delivery of a functional gene by tracheal instillation. A
Boulikas: An overview on gene therapy
116
drawback for retrovirus-mediated gene transfer arises from
that the majority of alveolar and airway epithelial cells are
terminally differentiated and only a small fraction of these
cells are proliferating and amenable to recombinant
retrovirus infection; however, lung epithelial cells are
prone to adenovirus infections because host cell
replication is not required for expression of adenoviral
proteins (see Rosenfeld et al, 1991).
The adenovirus major late promoter was linked to a
human α1-antitrypsin gene for its transfer to lung epithelia
of cotton rat respiratory pathway as a model for the
treatment of α1-antitrypsin deficiency; the cotton rat is an
animal commonly used to evaluate the pathogenesis of
adenoviral respiratory tract infections. Both in vitro and in
vivo infections have shown production and secretion of
α1-antitrypsin by the lung cells: cells, removed by
brushing the epithelial surface of the tracheobronchial tree
from the lungs of cotton rats demonstrated the possibility
of their infection in culture and secretion of the therapeutic
protein; following direct infection of the animals in vivo,
demonstrated that the protein was synthesized and
secreted in the epithelial lining fluid of the lung for over 1
week (Rosenfeld et al, 1991).
i.v. administration of a full-length cDNA encoding
human α1-antitrypsin (100 ng/mouse) encapsulated in
small liposomes resulted in expression in liver
parenchymal cells as shown on immunohistochemical
liver sections; this effect remained for at least 2 weeks and
some enzyme could be detected in plasma (Aliño et al,
1996). The human α1-antitrypsin gene was transferred to
lungs of rabbits; immunohistochemical staining showed
α1-AT protein in the pulmonary endothelium following
intravenous administration, in alveolar epithelial cells
following aerosol administration, and in the airway
epithelium by either route (Canonico et al, 1994). A
recombinant adenovirus vector was also used for α1-AT
cDNA transfer (Gilardi et al, 1990). α1-AT cDNA in an
adenoviral vector was administered by retrograde ductal
instillation to the submandibular glands of male rats;
transient expression took place in salivary glands (Kagami
et al, 1996).
Guo et al (1996) have evaluated the transcriptional
activities of 5 viral and cellular enhancer/promoter
elements, showing either high-level or hepatocyte-specific
expression following transient transfection into hepatoma
cells using recombinant adenoviruses expressing human
α1-antitrypsin; the human elongation factor 1α gene
promoter produced 2 µg/ml serum level of human α1antitrypsin,
which is physiologic in humans and will be
therapeutic for patients with α1-antitrypsin deficiency.
XXXVIII. Approaches to the gene
therapy of Parkinson’s disease (PD)