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

摘要

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.