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

intake when administered to adrenalectomized rats
(Zakrzewska et al, 1997).
D. Therapy of obesity with leptin infusion
High leptin levels are observed in obese humans and
rodents, suggesting that, in some cases, obesity is the
result of leptin insensitivity. To test this hypothesis Halaas
et al (1997) have used subcutaneous infusion of leptin to
lean mice; this resulted in a dose-dependent loss of body
weight at physiologic plasma levels. Chronic infusions of
leptin intracerebroventricularly (i.c.v.) at doses of 3 ng/hr
or greater resulted in complete depletion of visible adipose
tissue, which was maintained throughout 30 days of
continuous i.c.v. infusion. Direct measurement of energy
balance indicated that leptin treatment prevented the
energy decrease that follows reduced food intake but did
not increase total energy expenditure (Halaas et al, 1997).
In New Zealand Obese (NZO) mice, which were
unresponsive to peripheral leptin but were responsive to
i.c.v. leptin, obesity was the result of leptin resistance
most likely arising from a decreased transport of leptin
into the cerebrospinal fluid(Halaas et al, 1997).
Leptin administration reduced obesity in leptindeficient
ob/ob mice. Van Heek et al (1997) examined
whether diet-induced obesity in mice produces resistance
to peripheral and/or central leptin treatment. In a dietinduced
obesity model, mice exhibited resistance to
peripherally administered leptin, while retaining
sensitivity to centrally administered leptin (by a single
intracerebroventricular infusion). Whereas C57BL/6 mice
initially responded to peripherally-administered leptin
with a marked decrease in food intake, leptin resistance
developed after 16 days on high fat diet; however, central
administration of leptin to peripherally leptin-resistant
mice resulted in a robust response to leptin. Thus, the
effects of additional leptin administration in obese humans
who have high circulating leptin levels, especially after
intravenous injection (peripheral) versus
intracerebroventricular infusion, remain to be determined.
This study also implies the importance of the tissue target
for the delivery of the leptin gene for treatment of obesity
in humans.
E. The leptin and leptin receptor genes
Cloning of the gene encoding leptin (ob gene) and its
receptor (db gene) has provided spectacular insights in
elucidating the mechanisms involved in the control of food
intake and body weight maintenance in obese and lean
individuals. Transgenic mice lacking both alleles of either
ob or db genes showed early onset obesity from excessive
food intake and decreased energy expenditure, and in
addition showed severe insulin resistance, diabetes, and
sterility; administration of recombinant leptin had weight
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reducing effects (Campfield et al, 1995; Halaas et al,
1995; Pellymounter et al, 1995).
However, the vast majority of obese humans appear to
have excessively high levels of leptin and absence of
mutations in the OB gene (Considine et al, 1995; Maffei et
al, 1996). The nonsense mutation in the ob mouse which
results in the conversion of arginine 105 to a stop codon of
leptin gene was not present in human obesity. The defect
in humans is localized in the signaling pathway in the
brain which might involve: (i) the leptin receptor; (ii) the
Tub protein, expressed in the hypothalamus, that mediates
the signaling to the interior of the hypothalamus cell; (iii)
the Agouti protein which is expressed in all tissues in
obese mice but in the skin in normal mice, supposed to
antagonize melanocortin signal to the CNS; and (iv)
carboxypeptidase E (product of the fat gene) expressed in
endocrine and neuroendocrine tissues (reviewed by
Spiegelman and Flier, 1996).
Using a reverse transcription PCR product of the
coding region of the Obese (ob) gene from five lean and
five obese subjects it was determined that there was 72%
more ob gene expression in eight obese subjects compared
to eight lean controls; thus, ob gene expression is
increased in human obesity (Considine et al, 1995).
Limitation in food intake reduced the reproductive
competence, reduced the levels of thyroid hormone, and
activated the adrenal-pituitary stress axis; these starvation
adaptations were reversed with administration of
recombinant leptin (Ahima et al, 1996).
Obesity can result from a promotion in adipocyte
differentiation. A number of mitogens (PDGF, EGF, FGF,
tumor promoters), cytokines (TNF-α, IL-1, IL-6, TGF-β,
IFN-γ) and oncogenes inhibit adipocyte differentiation
and, therefore, inhibited adipogenesis and obesity. Insulin
plays a positive role in the differentiation of adipocyte
precursors and stimulates lipogenesis in adipose cells but
exerts a negative lipogenic response in fibroblasts;
preadipocytes which express small amounts of insulin
receptors require insulin or insulin-like growth factor-1 for
optimal differentiation.
The mechanism of inhibition of adipogenesis by
mitogens involves activation of the mitogen-induced MAP
kinase which phosphorylates at serine-112 the adipogenic
transcription factor PPARγ (peroxisome proliferatoractivated
receptor γ). PPARγ acts as a dimer with RXRα
to regulate adipocyte differentiation and sensitivity of the
adipose cells to insulin. PPARγ is the high affinity
receptor for the thiazolidinedione class of insulinsensitizing
drugs and the PPARγ-drug binding results in a
powerful adipogenic response; thus, factors which
stimulate the MAP kinase phosphorylation of PPARγ,
could cause resistance to insulin (Hu et al, 1996).
F. Gene transfer of the leptin gene