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

five GAL4 response elements, normally absent from
mammalian genomes; (ii) the synthetic hybrid steroid
receptor (TAXI), composed of the GAL4 DNA -binding
domain, a truncated human progesterone receptor, and the
acidic region from VP16 protein of HSV; the hybrid
molecule activates transcription from the UAS promoter
when bound to an inducer drug, and (iii) the synthetic
nontoxic drug inducer RU486 which is permeable to
blood-brain and placental barriers; this model allows up to
100-fold induction of a gene linked to this system and can
be finely tuned to lower levels of induction (Delort and
Capecchi, 1996).
Transient cotransfection of HeLa cells with the UAS-
CAT and the hybrid receptor expression vector showed
that the hybrid TAXI protein bound to the UAS promoter
only after treatment with RU486 but not progesterone; the
TAXI/UAS system was successfully used in transgenic
mice to regulate the expression of a human growth
hormone gene; the ex vivo approach, however, did not
sustain long-term expression of the transgene. This system
might allow physicians to alter the level of expression of
foreign genes during somatic cell transfer in response to
the clinical state of the patient (Delort and Capecchi,
1996).
Iida et al (1996) have modified the tetracyclinecontrolled
inducible system by the addition of the ligandbinding
domain of the estrogen receptor to the carboxy
terminus of the tTA transactivator; a single retroviral
vector could transduce both the transactivator gene and the
gene of interest controlled by the tTA-inducible promoter
into mammalian cells; cell lines expressing the
transactivator were established where the expression of a
gene (the toxic G protein of vesicular stomatitis virus)
depended on the removal of tetracycline and the addition
of estrogen.
A different genetic switch used consisted of the
cytochrome P450 1A1 promoter driving the expression of
the human apolipoprotein E (apoE) gene in transgenic
mice; this switch system was induced by βnaphthoflavone;
the inducer could pass transplacentally
and via breast milk from an injected mother to her
suckling neonatal pups, giving rise to the induction of
human apoE in neonate plasma and lowering the
cholesterol levels in hypercholesterolemic pups (Smith et
al, 1995).
XII. DNA recombination in gene
therapy
A. Mechanisms of DNA recombination
Genetic recombination, i.e., exchange of segments of
DNA between two molecules of DNA, is a very frequent
event. It often occurs during meiosis and also between
homologous chromosomes in mitosis. Homologous
recombination involving double-strand DNA breaks
(DSBs), has similarities to mechanisms of repair of DSB
Boulikas: An overview on gene therapy
36
lesions by cells. Specific recombinases have played and
continue to play an important role in molecular evolution
and genome shuffling; deregulation in recombination
procecess is connected to chromosomal aberrations
(inversions, translocations) in cancer. The double-strandbreak
repair model was put forward by Szostak and
collaborators (1983) to explain genetic recombination in
yeast. Recent studies (reviewed by Stahl, 1996) have
isolated the recombination intermediate molecules
predicted by the DSB repair model; in this model, a 5’-3’
exonuclease is responsible for the removal of segments of
single strands starting bidirectionally from the DSB
followed by invasion, repair synthesis and ligation to give
the joint molecule which is then reduced to a pair of
duplexes by a Holliday junction resolvase.
The development of mature lymphocytes in mammals
results from a complex combination of genetically
preprogrammed events and interactions with antigens.
Shared in its general mechanisms by both B (bone
marrow) and T (thymus) lymphocytes this developmental
program involves a series of cell migration gene
rearrangements, cell-to-cell contacts, as well as positive
and negative selection processes; recombination
mechanisms take place at the immunoglobulin and the T
cell receptor genes to generate a large number of
immunoglobulin genes in different lymphocyte clones.
One site-specific recombination event brings together the
V and the J segments of the light chain immunoglobulin
genes. In the case of the heavy chain genes, one
recombination event joins a V to a D segment,
sequentially followed in a time frame by the joining of the
recombined V-D segment to a J segment. Recent studies
have shown that the mechanism of V(D)J recombination is
a two-step process involving: (i) site-specific DNA
cleavage at the 7mer sequence and at the first nucleotide
of the coding sequence, implicating the RAG-1 and RAG-
2 proteins which are necessary and sufficient for this step
(van Gent et al, 1996); (ii) joining of broken ends in a
mechanism similar to the repair of double strand breaks.
The murine SCID locus has provided crucial information
in the elucidation of the second step in V(D)J
recombination: thymocytes in SCID mice are able to
catalyze joining of signal ends but display an
accumulation of hairpin coding ends (Zhu et al, 1996).
The murine SCID locus has been mapped to the gene
encoding the catalytic subunit of DNA-dependent protein
kinase (DNA-PK) (Kirchgessner et al, 1995).
Group I introns from a variety of organisms contain
long open reading frames (ORFs) that encode site-specific
DNA endonucleases which promote integration of their
DNA into cognate sites via homologous recombination.
These endonucleases typically cleave intron-lacking DNA
near the site of intron insertion (exon-exon junction)
creating a staggered DSB which facilitates intron invasion
(intron homing). This mechanism has been demonstrated
in mitochondria, chloroplasts and nuclei of eukaryotic