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

Gene Therapy and Molecular Biology Vol 1, page 119
Figure 34. Transfer of the tyrosine hydroxylase gene to primary muscle cells followed by transplantation of these cells to brains of THdeficient
rats has alleviated the number of collateral rotations of the animals which are models for Parkinson’s disease. Adapted from
Jiao et al, 1993. Reproduced from Boulikas T (1996b) Gene therapy to human diseases: ex vivo and in vivo studies. Int J Oncol 9,
1239-1251. With the kind permission from the International Journal of Oncology.
Implantation of immortalized rat fibroblasts releasing
L-dopa into the cell culture medium (Wolff et al, 1989), of
primary fibroblasts (Fisher et al, 1991) and myoblasts
(Jiao et al, 1993), stably transfected in culture with the TH
gene, reduced behavioral abnormalities in PD animal
models and the number of contralateral rotation dropped to
4 rotations/min (Figure 34).
Direct injection of lipofectin-plasmid DNA complexes
containing the TH gene under the influence of the SV40
promoter/enhancer (pSVK3 plasmid of Pharmacia) has
also shown expression of TH into striatal cells
compensating for the loss of the intrinsic striatal
dopaminergic input reducing quickly and significantly the
rotational abnormalities in rat models (Cao et al, 1995).
A different approach has been aimed at converting
endogenous striatal cells into L-dopa-producing cells; this
was obtained by infection of 6-hydroxydopamine-lesioned
rats, used as a model of PD, with a defective herpes
simplex virus type 1 vector expressing TH (During et al,
1994).
Recombinant adenovirus are attractive delivery
vehicles of genes to alleviate PD symptoms because they
can transduce both quiescent and actively dividing cells,
thereby allowing both direct in vivo gene transfer and ex
vivo gene transfer to neural cells; because the brain is
partially protected from the immune system, the
expression of adenoviral vectors can persist for several
months with little inflammation (reviewed by Horellou
and Mallet, 1997).
3. Glial cell line-derived neurotrophic factor
The rat glial cell line-derived neurotrophic factor
(rGDNF), a putative central nervous system dopaminergic
survival factor, was evaluated for its ability to protect
nigral dopaminergic neurons in the progressive Sauer and
Oertel 6-hydroxydopamine (6-OHDA) lesion model of
Parkinson's disease. Perinigral injections to rats of rGDNF
protected a significant number of cells when compared
with cell counts of rats injected with a recombinant AAV
carrying the lacZ gene (94% vs. 51%, respectively); this
treatment gave 85% of tyrosine hydroxylase-positive cells
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(vs. only 49% in the lacZ group) (Mandel et al, 1997; see
also Bohn and Choi-Lundberg, 1998, this volume).
XXXIX. Gene therapy of hemophilia A
and B
A. Gene therapy of hemophilia A
Hemophilia A, characterized by hemorrhagic episodes
of which the spontaneous intracranial bleeding could
result in crippling or death, affects 1 in 10,000 males. It is
caused by a deficiency in Factor VIII (FVIII), crucial in
blood coagulation, responsible for accelerating activation
of factor X by factor IXa in the presence of calcium and
phospholipids. Human FVIII is synthesized as a 2351amino
acid precursor protein with a 19- amino acid signal
peptide; the 256 kDa single-chain protein composed of the
homologous domains A1-A2-B-A3-C1-C2 is processed by
proteolysis to a heterodimer composed of a heavy chain
(90-200 kDa) and a light chain (80 kDa) which circulates
in plasma.
Both the 186 kb gene encoding human FVIII and the
7.2 kb cDNA sequences are known; recombinant FVIII
has been expressed from both intact cDNA and a cDNA
lacking the B domain; however, the expression of the
protein from cell culture was 100-1000 times lower than
the expression of other recombinant proteins. This is due
to the large size of the protein, its required proteolytic
processing, instability of mRNA, abnormal secretion from
inefficient transport from the endoplasmic reticulum to the
Golgi, and the required N- and O-linked glycosylation for
biological activity. Recombinant FVIII is being
administered to approximately 50% of the patients.
Biologically active FVIII has been produced recently in
the milk of transgenic pigs by targeting expression of
human FVIII cDNA to the mammary gland of the animals;
the expression of the transgene was driven by regulatory
sequences from the mouse whey acidic protein gene
(Paleyanda et al, 1997).
Infusion of purified factor VIII is the most widely used
therapy; however, protein replacement suffers from
transfusion-associated complications (AIDS and hepatitis
B and C infections); over 50% of the patients treated from