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

transduced primary mouse fibroblasts into the peritoneal
cavity in SCID mice resulted in correction of hemophilia
A (Dwarki et al, 1995). Ex vivo transduction of primary
myoblasts in mice with the factor IX gene followed by
transplantation of the transduced cells led to partial
correction of hemophilia B (Dai et al, 1992; Yao et al,
1994).
Intraarticular injection of syngeneic synovial cells
transduced with the IL-1R antagonist protein gene
alleviated the symptoms of arthritis (Bandara et al, 1993).
Similarly, ex vivo retroviral transfer of the secreted human
IL-1Ra cDNA to primary synoviocytes followed by
engraftment in ankle joints of rats with induced arthritis
significantly suppressed the severity of the disease
(Makarov et al, 1996; see also Ghivizzani et al, 1997). To
demonstrate feasibility of the ex vivo FH therapy, three
baboons underwent a partial hepatectomy, their
hepatocytes were isolated, cultured, transduced with a
retrovirus containing the human LDL-R gene, and infused
via a catheter (Grossman et al, 1992).
An important number of studies on cancer
immunotherapy have been performed on animal models
(For example, see Vieweg et al, 1994; Wiltrout et al, 1995;
Caruso et al, 1996; Bramson et al, 1996; Tahara et al,
1996; Zhang et al, 1996; Rakhmilevich et al, 1997; Aruga
et al, 1997; Clary et al, 1997; Ju et al, 1997)
Studies with tumor cells reconstituted with RB ex vivo
and implanted into immunodeficient mice have
demonstrated cancer suppression (see Riley et al, 1996).
Transfer of the Cu 2+
/Zn 2+
superoxide dismutase into ex
vivo modified cells protected the cells from oxidative
damage during manipulation and increased their survival
after implantation (Nakao et al, 1995). Ex vivo transfer of
the MDR1 gene in bone marrow cells has been used to
render stem cells resistant to cancer chemotherapy (Lee et
al, 1998, this volume). Ex vivo transduction of MCF-7
human breast cancer cells with antisense c-fos produced
expression of antifos RNA, and inhibited s.c. tumor
growth and invasiveness in breast cancer xenografts in
nude mice (Arteaga and Holt, 1996).
Direct in vivo injection of a gene (intratumoral,
intravenous, etc) must be distinguished from ex vivo gene
therapy methods. Some representative direct in vivo
studies to animals using genes are summarized on Table
6.
B. Ex vivo gene therapy on humans
The first person to be treated ex vivo was a 4-year-old
suffering with ADA deficiency in 1990 (see ADA
deficiency below). The US Patent Office has issued in
1995 a patent covering all ex vivo gene therapy to French
Anderson, Steven Rosenberg, and Michael Blaese; the
technique was developed at NIH in the 1980s and an
exclusive license to this work has been awarded to Gene
Gene Therapy and Molecular Biology Vol 1, page 87
87
Therapy Inc, (Rockville, Maryland). Of the 220 protocols
for Clinical Trials approved by NIH's Recombinant DNA
Advisory Committee (RAC), a significant number (over
100) use ex vivo gene therapy applications (see Gavaghan,
1995). Ex vivo protocols are marked In Vitro in
Appendicx 1 and Table 4 in following article (pages 203-
206). Also protocols proposing immunotherapy use ex
vivo transduction of cells from cancer patients with
cytokine genes and immunization of the patient with the
transduced cells (Appendix 1).
Transduction of cells in vitro with adenoviruses makes
the patients own cells antigenic leading to their destruction
by T lymphocytes thus eliminating the therapeutic effect
after reimplantation (e.g. Yang et al, 1994). It was thought
that this antigenicity arises from the adenoviral proteins
expressed in transduced cells; however, recent data have
demonstrated that antigenicity could also arise from the
expression of the therapeutic recombinant protein (see
above).
Ex vivo approaches have concentrated on correction of
mutated genes involved in purine metabolism including
adenosine deaminase (ADA) deficiency in severe
combined immunodeficiency (SCID) patients, PNP
(purine nucleoside phosphorylase) deficiency, and the
therapy of Lesh-Nyhan syndrome caused by a deficiency
in hypoxanthine-guanine phosphoribosyltransferase (HG-
PRT). The first human trial to be approved for ex vivo
gene therapy was for the treatment of ADA deficiency
which began in 1990 (Karlsson, 1991; Ferrari et al, 1991).
Ex vivo studies include transfer of factor IX gene in skin
fibroblasts from hemophilia B patients in China followed
by subcutaneous injection of the cells to the patient
(Wilson et al, 1992; reviewed by Anderson, 1992). From
1990-1992, a clinical trial was initiated using retrovirus
mediated transfer of the 1.5 kb ADA gene cDNA to T
cells from two children with severe combined
immunodeficiency following multiple transplantations of
ex vivo modified blood cells; the vector was integrated and
the ADA gene was expressed for long periods (Blaese et
al, 1995; Bordignon et al, 1995).
A clinical protocol for the therapy of amyotrophic
lateral sclerosis uses a semipermeable membrane to
enclose the ex vivo modified xenogenic BKH cells; the
membrane is implanted intrathecally to provide human
ciliary neurotrophic factor (Deglon et al, 1996; Pochon et
al, 1996). An ex vivo clinical trial on humans,
homozygous for mutations in the LDL receptor gene, is
performed using cultured hepatocytes from the patient
which are transduced ex vivo with the LDL receptor gene
and transplanted by infusion into the portal vein of the
patient (Wilson et al, 1992; Grossman et al, 1994).
Cancer immunotherapy uses transfer of cytokine genes
(IL-2, IL-7, IFN-γ, GM-CSF) to autologous (cancer
patient’s) cells followed by immunization of the patient to
elicit activation of tumour-specific T lymphocytes capable