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

1977 to 1985 were infected with HIV. Improved
manufacturing procedures and production of recombinant
factor VIII have reduced infectious complications.
Hemophilia A is particularly amenable to gene therapy
because even a slight increase in blood plasma of factor
VIII can convert a severe form of the disease to a mild
form. Application of retrovirus-mediated transfer of factor
VIII gene has been hampered by a 100- to 1000-fold
reduction in mRNA accumulation and protein production
in cells, as well as in retrovirus titer, because of the
presence of a secondary structure (arising from inverted
repeats) within the DNA coding region of clotting factor
VIII gene (Lynch et al, 1993; Chuah et al, 1995). Partial
solution to this problem has been provided by Chuag and
coworkers (1995) who determined that insertion of a 5'
intron in the retrovirus vector increased 20-fold gene
expression and 40-fold virus titer after transfection of the
human T cell line SupT1, human Raji Burkitt B
lymphoblastoma and other cell lines.
An more exciting approach has been the use of a Bdomain-deleted
form of factor VIII; at the protein level,
this domain is not required for pro-coagulant activity and
retains the thrombin-cleavage sites. Transferrin-mediated
transfection of fibroblasts and myoblasts with B-domaindeleted
factor VIII gene followed by implantation into
mice gave therapeutic levels of factor VIII in the blood of
the animals for 24 hours (Zatloukal et al, 1994).
A similar ex vivo transfection procedure used retroviral
vectors for the introduction of B-domain−deleted factor
VIII gene into primary mouse fibroblasts in culture; use of
the MFG vector, which utilizes authentic viral splicing
signals and lacks a selectable marker gene was crucial in
producing high titer viral stocks (Dwarki et al, 1995). This
procedure was followed by surgical implantation into the
peritoneal cavity in SCID mice of 15 million cells in the
form of neo-organs formed on expanded poly(tetra
fluoroethylene) (PTFE) fibers after they were coated with
type I collagen from rat tail. Coated fibers were arranged
to the bottom of tissue culture dishes and genetically
modified cells were allowed to solidify on the PTFE fibers
for 1-2 days; the levels of factor VIII obtained were 50-
1000 ng/ml which are 10-fold higher than those required
for correction of hemophilia A (Dwarki et al, 1995).
Efficient delivery of factor VIII was also observed after
direct i.v. or i.p. injection but not after i.m. or s.c. injection
of genetically-modified cells; this difference might arise
from protease levels in the extracellular space of muscle
and skin (Dwarki et al, 1995).
The subject is being reviewed in depth by Connelly
and Kaleko (1998) and Hoeben (1998) in this volume.
B. Gene therapy of hemophilia B
Hemophilia B is caused by a defect in the blood
clotting factor IX (FIX) affecting about 1 in 30,000 males.
Boulikas: An overview on gene therapy
120
The therapy consists on administration of factor IX
concentrates prepared from human plasma, a fact that led
to the infection of hemophiliacs with HIV and hepatitis B
virus in the 80s. Current research efforts are focused on
the delivery of factor IX gene using ex vivo transduction
of primary myoblasts in mice with factor IX gene
followed by transplantation of the transduced cells (Dai et
al, 1992; Yao et al, 1994). Mouse primary myoblasts were
infected with retrovirus expressing the canine factor IX
under control of mouse muscle creatine kinase and human
CMV promoter; successfully infected myoblasts, selected
in the presence of G418, were injected into the hindlegs of
recipient mice; secreted canine factor IX was monitored in
the plasma. Sustained expression of factor IX for over six
months without any apparent adverse effects on the
recipient mice was obtained; however, the levels of the
factor IX protein secreted into the plasma (10 ng/ml for
10 7
injected cells) were not sufficient to be of therapeutic
value but 100 times below the desired levels (Dai et al,
1992).
Dogs, lacking a functional factor IX gene, have been
used as animal models for hemophilia B. The liver of the
animals is the organ responsible for the production of
factor IX; direct infusion of recombinant retroviral
vectors, carrying the canine factor IX gene, into the portal
vein cannulated into a splenic vein in animals previously
subject to two-thirds hepatectomy resulted in the
expression of low levels of factor IX for up to about 5
months; about 0.3-1% of hepatocytes were found to be
transduced and stained blue with X-Gal in liver sections
when the β-galactosidase gene of E. coli was delivered
with the same retroviral vector (Kay et al, 1993).
A sustained partial correction of the defect was
succeeded in hemophilia B dogs by directly delivering the
factor IX gene in adenovirus vectors by injection into the
splenic veins of 1.6 to 2.2 pfu/Kg adenovirus (Kay et al,
1994). The therapy, however, obtained was transient and
although these animals displayed 300% factor IX levels in
their blood at day 2 from treatment, the levels dropped to
1% by three weeks and to 0.1% by 2 months (Kay et al,
1994). Since T cell immunity was responsible for
attacking and eliminating virally-transduced cells from the
body, clearing the corrected cells from the liver of
animals, daily administration of 19.5 mg/Kg cyclosporin
A led to prolongation of the therapeutic effect and to the
persistence of adenovirus-transduced cells (10% of factor
IX levels by two months (Fang et al, 1995; Figure 35).
Cyclosporin A (CsA) is a cyclic peptide, fungal
metabolite, displaying low myelotoxicity but toxic to T
cell lymphocytes; it is widely used for immunosuppression
of individuals receiving renal and other organ
transplants but also for the therapy of autoimmune
diseases. CsA inhibits activation of IL-2 gene, an early
event required for T cell activation and this mechanism is
thought to govern its immunosuppressive effects.