Vector development for suicide gene therapy
Author: Aints, Alar
Date: 2002-05-24
Location: Föreläsningssalen, Rehabgatan 64 (R64), Huddinge Universitetssjukhus
Time: 9.00
Department: Institutionen för medicin / Department of Medicine
View/ Open:
thesis.pdf (1.295Mb)
Abstract
Gene therapy is used to treat conditions that arise from errors in the
genetic makeup of cells either congenital diseases resulting from a
deletion or mutation in a gene or malignant diseases where genetic
regulation mechanisms have been deranged.
Suicide gene therapy is one of several gene therapeutic approaches to
treat cancer. A suicide gene is a gene encoding a protein, frequently an
enzyme, that in itself is non-toxic to the genetically modified cell.
However, when the cell is exposed to a specific non-toxic prodrug, this
is selectively converted by the gene product into toxic metabolites that
kill the cell.
The gene transfer technologies available today have limited effectiveness
- only a fraction of target cells can be genetically modified. This can
be compensated by several processes taking place after transgene
expression and enzymatic conversion of the prodrug. In vivo, and also in
cell culture, gap junction mediated intercellular communication (GJIC)
enables the spread the converted metabolites. However, many tumours
lacking connexin expression are not susceptible to this form of
treatment.
To bypass the requirement for gap junctions, one can link the HSV-tk gene
to the gene of another herpes virus protein, VP22. The VP22 protein has
been shown to pass freely between cells. We show the ability of VP22-GFP
fusion protein to spread in cell culture and translocate into the nucleus
of the recipient cells (Paper I), Also, VP22 fusion to TK (VP22-TK) is
transferred to the surrounding cells in cell culture and in vivo in a
mouse tumour model, in the absence of gap junction formation in a
connexin negative tumour cell line and sensitises the tumours to GCV.
However, relatively high proportion (50%) of VP22-TK expressing cells is
necessary to exert a full effect (Paper II). Therefore, we have analysed
the VP22 protein in detail. Mapping of functional domains revealed
several independent functional domains and localisation signals in the
VP22 polypeptide. These constructs can have a potential use in targeting
defined functional peptides to specific subcellular locations (Paper
III).
Suicide gene technology can provide a safety switch for cytotoxic
effector cells, to treat graft versus host disease (GvHD). In this
application, it would be ideal to achieve 100% effectiveness of
modification of the target cells. Different selection marker genes for
selection have been utilised, and these fall into two categories:
metabolic and physical selection markers. As there is requirement for a
resistance marker better suited for selection of human cells, we have
characterised the ouabain resistance gene (OuaR).
OuaR belongs to the Na+ ,K+ -ATPase gene family - a housekeeping enzyme
present in all mammalian cells. It maintains cellular homeostasis of Na+
and K+. Ouabain belongs to cardiac glucoside group of drugs. It
inactivates the naturally occurring non-resistant Na+ ,K+ -ATPase which
results in very rapid cell death. OuaR is a PCR-generated point mutant
L799C that is completely resistant to inhibition by ouabain and can be
used for background free selection in very short time - 12-36 hours,
compared to 10- 15 days for classical antibiotic selection. We show the
versatility of OuaR gene transfer by transient transfection and stable
retrovirus-mediated integration in several cultured cell lines as well as
primary human donor T-lymphocytes (Paper IV).
We have prepared suicide gene therapy vectors containing HSV-tk and OuaR
selection marker fusion gene in extensively optimised retroviral backbone
SF91. The OuaR selection marker allows the transduced cells to be
selected chemically in 36 hours, reducing the necessary time for in vitro
culture to a week. The transduced T-cells display high sensitivity to
gancyclovir. The rapid gene transfer and selection process prevents
culture-related changes in T-cell function and helps to establish
protocols to gain control over cytotoxic cells by suicide gene transfer
(Paper V).
In conclusion, we have characterised vectors for suicide gene therapy
with the potential to achieve 100% efficiency of genetic modification of
the target cells. VP22 protein vectors enable the spread of fusion
protein to all surrounding cells after gene expression in the
gene-modified cells. Viral or plasmid vectors containing the OuaR
selection marker allow the gene- modified cells to be selected
immediately after gene expression, reducing the time for selection and
time-dependent in vitro cell culture-related sideeffects to minimum.
List of papers:
I. Aints A, Dilber MS, Smith CI (1999). "Intercellular spread of GFP-VP22. " J Gene Med 1(4): 275-9
Pubmed
II. Dilber MS, Phelan A, Aints A, Mohamed AJ, Elliott G, Smith CI, OHare P (1999). "Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22. " Gene Ther 6(1): 12-21
Pubmed
III. Aints A, Guven H, Gahrton G, Smith CI, Dilber MS (2001). "Mapping of herpes simplex virus-1 VP22 functional domains for inter- and subcellular protein targeting. " Gene Ther 8(14): 1051-6
Pubmed
IV. Aints A, Belusa R, Andersson RM, Guven H, Dilber MS (2002). "Enhanced ouabain resistance gene as a eukaryotic selection marker. " Hum Gene Ther 13(8): 969-77 (In Print)
Pubmed
V. Aints A, Unger C, Rabbani H, Dilber MS (2002). "Rapid suicide gene transfer and selection of primary human T-cells." (Manuscript)
I. Aints A, Dilber MS, Smith CI (1999). "Intercellular spread of GFP-VP22. " J Gene Med 1(4): 275-9
Pubmed
II. Dilber MS, Phelan A, Aints A, Mohamed AJ, Elliott G, Smith CI, OHare P (1999). "Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22. " Gene Ther 6(1): 12-21
Pubmed
III. Aints A, Guven H, Gahrton G, Smith CI, Dilber MS (2001). "Mapping of herpes simplex virus-1 VP22 functional domains for inter- and subcellular protein targeting. " Gene Ther 8(14): 1051-6
Pubmed
IV. Aints A, Belusa R, Andersson RM, Guven H, Dilber MS (2002). "Enhanced ouabain resistance gene as a eukaryotic selection marker. " Hum Gene Ther 13(8): 969-77 (In Print)
Pubmed
V. Aints A, Unger C, Rabbani H, Dilber MS (2002). "Rapid suicide gene transfer and selection of primary human T-cells." (Manuscript)
Issue date: 2002-05-03
Rights:
Publication year: 2002
ISBN: 91-7349-199-3
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