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

B. Gene therapy of FH: experiments in cell
culture
A transmissible retroviral vector containing a fulllength
human cDNA for the LDL-R was used to infect
fibroblasts from the Watanabe heritable hyperlipidemic
(WHHL) rabbit which expressed the human receptor
efficiently, as indicated by RNA and ligand blotting
studies (Miyanohara et al, 1988). The number of
hepatocytes that could be transduced by retroviruses
bearing the therapeutic gene was one of the limiting steps
that could impair the success of this strategy; addition of
human hepatocyte growth factor (HGF) to hepatocytes
allowed marked increase in the transduction efficiency in
mouse (up to 80%) and human (40%) hepatocytes (Pages
et al, 1995). Transduction of the human LDL-R cDNA
under the transcriptional control of the liver-type pyruvate
kinase promoter allowed high and tissue specific
expression of the gene in primary hepatocytes; a second
vector with a housekeeping promoter corrected the LDL-R
deficiency in fibroblasts from a FH patient (Pages et al,
1996b).
C. Gene therapy of FH: experiments on
animals
Liver is the preferred target organ for gene transfermediated
treatment of FH. The presence of unique
receptors at the cellular membrane of hepatocytes forms
the basis for transfer strategies based on receptor targeting
(reviewed by Sandig and Strauss, 1996). An authentic
animal model used in FH gene therapy is the Watanabe
heritable hyperlipidemic rabbit which is homozygous for
FH and has a deletion in a cysteine-rich region of the LDL
receptor gene; this renders the receptor completely
dysfunctional (Yamamoto et al, 1986); these animals
display high levels of serum cholesterol, diffuse
atherosclerosis, and die prematurely. Liver tissue was
removed from such animals and the cultured hepatocytes
were transduced with retroviruses carrying the rabbit LDL
receptor gene; the genetically corrected cells were
transplanted into the animal from which they were
derived. This treatment resulted in a 30-40% reduction in
serum cholesterol that lasted for at least 4 months
(Chowdhury et al, 1991).
The portal vein has been used for liver targeting in
New Zealand White (NZW) rabbits. Expression of lacZ
was obtained in virtually all hepatocytes within 3 days
(but was undetectable by 3 weeks) after transfer of the
lacZ reporter gene under the control of different promoters
using recombinant, replication-defective adenoviruses
which were infused into the portal circulation. An
adenovirus human LDL-R cDNA was then infused into
the portal vein of rabbits deficient in LDL receptor and
demonstrated human LDL receptor protein in the majority
of hepatocytes that exceeded the levels found in human
Boulikas: An overview on gene therapy
98
liver by at least 10-fold. Transgene expression diminished
to undetectable levels within 3 weeks (Kozarsky et al,
1994).
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 that had been placed into the inferior
mesenteric vein at the time of liver resection (Grossman et
al, 1992). Gene replacement therapy of human LDL
receptor gene into the murine model of FH transiently
corrected the dyslipidaemia; long-term expression of the
therapeutic gene was extinguished by humoral and cellular
immune responses to LDL receptor which developed and
possibly contributed to the associated hepatitis (Kozarsky
et al, 1996).
As an alternative strategy, expression in the liver of the
very low density lipoprotein (VLDL) receptor, which is
homologous to the LDL receptor but has a different
pattern of expression, using recombinant adenoviruses
corrected the dyslipidaemia in the FH mouse; transfer of
the VLDL receptor gene circumvented immune responses
to the transgene leading to a higher duration of metabolic
correction (Kozarsky et al, 1996).
Replication-defective adenovirus-mediated gene
transfer of the very low density lipoprotein receptor
(VLDL-R) driven by a cytomegalovirus promoter in LDL-
R knockout mice by a single intravenous injection resulted
in reduction in total cholesterol by approximately 50% at
days 4 and 9 and returned toward control values on day
21. Lowering in total cholesterol was mediated by a
marked reduction in the intermediate density
lipoprotein/low density lipoprotein (IDL/LDL) fraction. In
treated animals, there was also an approximately 30%
reduction in plasma apolipoprotein (apo) E accompanied
by a 90% fall in apoB-100 on day 4 of treatment. Thus,
adenovirus-mediated transfer of the VLDLR gene induced
high-level hepatic expression of the VLDL-R, resulted in a
reversal of the hypercho-lesterolemia, and enhanced the
ability of these animals to clear IDL (Kobayashi et al,
1996).
The human apolipoprotein E (apoE) gene driven by
the cytochrome P450 1A1 promoter produced transgenic
mice where robust expression of apoE depended upon
injection of the inducer β-naphthoflavone; a transgenic
line exhibiting basal expression of apoE in the absence of
the inducer upon breeding with hypercholesterolemic
apoE-deficient mice produced animals which were as
hypercholesterolemic as their nontransgenic apoEdeficient
littermates in the basal state. When injected with
the inducer, plasma cholesterol levels of the transgenic
mice decreased dramatically. The inducer could pass
transplacentally and via breast milk from an injected
mother to her suckling neonatal pups, giving rise to the