Development of a gene therapy approach for the treatment of human mammary carcinoma using tissue specific retroviral vectors

Metastatic breast cancer is commonly thought to be incurable, but
gene therapy strategies with suicide genes are emerging as a
potential treatment for metastatic breast cancer. Retroviral
vectors are attractive candidates for such in vivo gene therapy
applications. Most of these vectors contain viral promoters that
are not tissue specific. In order to specifically target malignant
cells while at the same time sparing normal tissue, cancer gene
therapy will need to combine highly selective delivery with highly
specific gene expression, specific gene product activity and,
possibly, specific drug activation. Use of cell type or tissue
specific promoters would allow specificity of gene expression to be
achieved. It is therefore important to identify and characterise
tissue/tumour specific promoter/enhancer elements in and
combination with retroviral vector systems. In this present study,
the ProCon system was used to construct tissue specific vectors
containing either the viral MMTV U3 region or the cellular WAP
proximal promoter. It was important to characterise the vector
system at the molecular level as well as investigating their
targeted expression in vitro and in vivo. Validation of this system
was first made with a hybrid MMTV-BAGgal vector. Here, it could be
shown that the U3 from the 3' LTR could be replaced with the viral
MMTV U3 region and that the hybrid vector behaves as expected at
the molecular level throughout the whole transduction cycle. In
transgenic mice, the ability of a small fragment of the WAP
promoter to direct mammary specific expression was shown and this
raised the possibility to replace the viral promoter with the WAP
NRE. The replacement of viral U3 from the 3' LTR with the cellular
WAP NRE showed no effect either at the molecular level or on the
transduction cycle. These hybrid vectors were produced at titres
ranging from 103 to 105 cfu/ml, showing that swapping of the U3 of
3' LTR with either heterologous viral or cellular sequences does
not have an adverse affect on titre. Following infection and
reverse transcription, the expression of reporter gene should be
under the control of either the WAP NRE or the MMTV U3 region. In
order to determine the activity of the WAP NRE and MMTV U3 in the
context of a retroviral vector in vitro, suitable cell culture
conditions were established for both primary breast tumour cells as
well as established tumour cell lines. Primary human breast tumour
cells and breast tumour or non-breast tumour cell lines were
infected in monolayer culture and were analysed in a three
dimensional cell culture system for expression of the reporter
gene. Here, it was demonstrated that the MMTV U3 was mainly active
in human breast tumours. The WAP NRE was also able to direct
heterologous gene expression in the context of a modified
retroviral vector in human breast tumour cell lines as well as in
primary cells in culture, but not in other types of human tumour
cells. The activity of the WAP NRE in human breast tumour cells was
not necessarily to be expected, particularly since a human WAP
homologue has yet to be identified. An in vivo approach involving
transplantation into SCID/bg mice allowed a determination of
whether or not the in vitro response is reflected in vivo and to
complement the in vitro studies. This work showed that mice did
indeed develop both tumours and metastases. Both the MMTV U3 and
WAP NRE in the context of the ProCon system behaved in a similar
manner both in vitro and in vivo. Transgenic mice were also made in
order to analyse the tissue specificity of the MMTV U3 region in
the context of the ProCon system. A recloned provirus DNA was used
for the establishment of the mice. Expression of the MMTVProCon was
mainly restricted to the mammary gland and spleen, mirroring the
expression pattern of MMTV. The ability of the WAP proximal
promoter and MMTV U3 region to function in the context of a
retroviral vector is of interest for potential use to drive
therapeutic gene expression in gene therapy strategies directed
against breast cancer. Such genes could include those encoding
toxic products, for example the diphtheria toxin gene or,
alternatively, suicide genes allowing drug induced cell death with
a bystander activity. Such strategies will be successful if ectopic
expression can be avoided. In this study it was demonstrated that
the proximal WAP promoter was more mammary tumour cell specific and
it is therfore more suitable for use in gene therapy at this time.
Whilst the MMTV U3 region, in the form used here, may exhibit
ectopic expression, further experiments may identify regions or
fragments that can be used without observing this phenomenon.
Further, these experiments could be extended and combined with
systematic delivery of hybrid retroviral vectors with a therapeutic
gene either locally or into the bloodstream of xenotransplanted
SCID/bg mice. In order to accomplish this, a high titre viral
supernatant will be required and therefore efficient production
systems will have to be developed and tested.