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

Gene Therapy and Molecular Biology Vol 1, page 39
Figure 15. Large T antigen-immortalized breast cancer cells change morphology and lose expression of T antigen after infection
with Cre-Puro retrovirus: after prolonged culture the cells were infected with Cre-Puro retrovirus (B, D) or mock-infected (A, C),
selected for Puromycin resistance (A, B, C, D) and resistance to ganciclovir (B, D) and analyzed by light microscopy (A, B) and staining
with a large T-antibody (C, D). From Li LP, Schlag PM, Blankenstein T (1997) Transient expression of SV 40 large T antigen by
Cre/LoxP-mediated site-specific deletion in primary human tumor cells. Hum Gene Ther 8, 1695-1700. Reproduced with the kind
permission of the authors and Mary Ann Liebert, Inc.
Reversible immortalization of primary cells was
achieved by Westerman and Leboulch (1996) using
retrovirus-mediated transfer of an oncogene that could be
subsequently excised by site-specific Cre/LoxP
recombination; the FLP/FRT recombination was not
efficient in primary cells. Pure populations of cells in
which the oncogene was permanently excised were
obtained which reverted to their preimmortalized state.
Using the Cre/LoxP recombination strategy primary cells
could be cultured and expanded; the method was proposed
to be applicable for facilitating gene transfer to cells
unresponsive to exogenous growth factors.
A retroviral vector, containing both a neomycin
resistance expression unit flanked by loxP sites and GM-
CSF cDNA, was used to transduce the human
hematopoietic K-562 cell line. Superinfection of K562 cell
clones with a retrovirus containing a Cre recombinase
expression unit and molecular analyses of 30 doubly
transduced subclones showed a strict correlation between
Cre expression and LoxP-flanked selectable cassette
excision; excision of the selectable cassette resulted in a
significant increase of GM-CSF transcription driven by
the retroviral promoter (Fernex et al, 1997).
Novel retroviral vectors for gene transfer were
developed by Bergemann et al (1995) by inserting two
LoxP sites into a retroviral vector also containing the
HSV-tk gene; Cre expression in cells infected with this
vector was followed by BrdU selection for cells in which
site-specific recombination took place. Furthermore,
replacement of the enhancer/promoter elements in both
LTRs by Lox sequences led to the development of
retroviral suicide vectors for gene therapy. Vanin et al
(1997) have used the Cre/LoxP recombinase system to
generate high-titer retroviral producer cell lines;
incorporation of LoxP sites at the flanks of a Neo R -HSV-tk
cassette in the proviral DNA allowed excision of these
selectable markers through expression of Cre recombinase
and the production of a high-titer producer cell line
containing a single LoxP site flanked by the viral LTRs.
Retransfection of this cell line with a plasmid containing a
gene of interest flanked by LoxP sites and the Cre
expression vector allowed insertional LoxP/LoxP
recombination of the gene into the favorable preexisting
site in the genome and the generation of a new line with a
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titer equivalent to that of the parental producer cell line.
The Cre/LoxP recombinase strategy has been used to
generate retroviral vectors with the ability to excise
themselves after inserting a gene into the genome (Russ et
al, 1996).
Bushman and Miller (1997) fused retroviral integrase
enzymes to sequence-specific DNA-binding domains and
investigated target site selection by the resulting proteins.
A fusion protein composed of HIV integrase linked to the
DNA-binding domain of λ repressor was able to direct
selective integration of retroviral cDNA in vitro into target
DNA containing λ repressor binding sites. A fusion of
HIV integrase to the DNA binding domain of the zinc
finger protein Zif268 also directed increased integration
near Zif268 recognition sites.
Introduction of foreign DNA into cell nuclei with
recombinase cDNA and appropriate sequences to promote
recombination may promote nor only insertion of a
therapeutic gene into a specific chromosomal site but also
chromosomal rearrangements that could convert
therapeutically transduced cells into malignant. There is a
great deal of knowledge to be derived from these very
promising strategies of gene therapy before they can be
successfully applied to humans.
XIII. Fate of the transgene in the
nucleus
A. How to sustain transgene expression?
A major drawback in gene therapy applications is loss
in gene activity within a few days from gene transfer
although all previous steps were successful. In other
words, the transferred gene is transiently expressed for 1-4
days and its expression thereafter declines dramatically.
This is due (i) to the degradation of the gene in the
nucleus; (ii) the dilution of the plasmid during replication
of the cells from its inability to replicate; (iii) its
inactivation by position effects from chromatin
surroundings after its integration into the chromosomal
DNA; (iv) the elimination of the therapeutic cells
expressing the transgene by the immune system of the
organism either because of the antigenicity of the
expressed protein or because of the antigenicity of viral