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

of human immunoglobulin and antigen-specific T cells)
(Ferrari et al, 1991). Ex vivo correction of the defect in T
cells from ADA-deficient patients with retroviral vectors
gave to these cells an advantage for cell division against a
background of slowly dividing uncorrected T cells after
their transplantation (Karlsson, 1991).
XXXVI. Gaucher disease, lysosomal
storage disease, and
mucopolysaccharidosis VII
Many mutations affecting the glucocerebrosidase gene
have been defined as causes of the glycolipid storage
disorder, Gaucher disease; disease symptoms are a result
of macrophage engorgement secondary to this enzyme
deficiency. The recombinant from of glucocerebrosidase
imiglucerase protein is effective in treating the disease as a
replacement therapy (reviewed by Beutler, 1997).
An amphotropic producer cell line that synthesized
viral particles carrying a fusion of the selectable MDR1
cDNA encoding P-glycoprotein (P-gp) and the human
glucocerebrosidase gene was constructed; complete
restoration of glucocerebrosidase deficiency in Gaucher
fibroblasts was achieved using this retrovirus; selection of
the transduced Gaucher fibroblasts in colchicine (MDR1
function) raised their glucocerebrosidase activity from
nearly undetectable to normal levels; combination of much
lower concentrations of colchicine and inhibitors of the Pgp
pump (verapamil) allowed to select for high-level
expression of MDR1 and glucocerebrosidase; this
regimen, in clinical use for the treatment of multidrugresistant
malignancies, may find application for high level
selection of a nonselectable gene such as
glucocerebrosidase (Aran et al, 1996; see also Migita et al,
1995).
Allogenic bone marrow transplantation (Parkman,
1986) or intravenous infusion of glucocerebrosidase
(enzyme replacement therapy) in a patient with Gaucher's
disease (Barton et al, 1990), although has partially
corrected deficiencies in lysosomal enzymes, was not
amenable to brain cells because of the brain barrier and
cannot alleviate symptoms in the central nervous system.
Protocols #38, 39, and 51 use glucocerebrosidase cDNA to
transduce CD34 + autologous peripheral blood cells
followed by intravenous injection of the transduced cells
into patients with Gaucher's disease. CD34 + cells obtained
from G-CSF mobilized peripheral blood stem cells or from
bone marrow (#51) are being transduced ex vivo and
reinfused into the patient at the National Institutes of
Health and Children’s Hospital of Los Angeles (Dunbar
and Kohn, 1996).
Type II Glycogen Storage Disease, a deficiency of
acid α-glucosidase (GAA), results in the abnormal
accumulation of glycogen in skeletal and cardiac muscle
lysosomes; this can have devastating effects ultimately
Gene Therapy and Molecular Biology Vol 1, page 115
115
leading to death; the wild-type enzyme was produced in
deficient myoblasts after gene transfer with a retroviral
vector carrying the cDNA for GAA. The transduced cells
secreted GAA that was endocytosed via the mannose-6phosphate
receptor into lysosomes of deficient cells and
digested glycogen; thus, the transduced cells provided
phenotypic correction to distant cells in the culture by
secretion. Figure 33 shows that the GAA-transduced
myoblasts (red fluorescence) were able to fuse with
deficient myoblasts (green fluorescence) and provide
enzyme activity mediating phenotypic correction to
neighboring GAA-deficient cells (Zaretsky et al, 1997).
Aspartylglucosaminuria (AGU, appearance of
aspartylglucosamine in the urine) is the only known
human disease caused by an amidase deficiency, in this
case by deficiency of the enzyme aspartylglucosaminidase
(AGA) in virtually all cell types of patients; AGA
deficiency fails to perform the final breakdown of
asparagine-linked glycoproteins leading to the intralysosomal
accumulation of uncleaved glycoasparagines and
to their abnormal urinary excretion. AGU patients display
progressive psychomotor retardation starting in early
childhood. The most common mutation in the Finnish
population responsible for AGA deficiency is a point
mutation resulting in the amino acid substitution Cys-163
to Ser in the AGA gene (Ikonen et al, 1991).
Retrovirus-mediated gene transfer was successfully
used to correct the AGA gene in cultured human primary
fibroblasts and lymphoblasts from AGU patients as a
prelude to ex vivo human gene therapy; enzyme correction