NFP37 SOMATIC GENE THERAPY Texts for Laypersons

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Dr. Walter Reith;	Génétique et Microbiologie; Centre Méd. Univ.; 9, avenue de Champel; 1211 Genève 4; CH; Tel 022 702 56 66; Fax 022 702 57 02; e-mail: reith@cmu.unige.ch
Title:	Construction of mouse models and regulatory systems designed to optimise the control of transgene expression.
Co-applicants:	Prof. JD Vassalli (Genève)
Collaborators:
	ABSTRACT | PUBLICATION | DIVULGATIONTEXT | BACK TO OUTLINE

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ABSTRACT FOR LAYPERSONS 1997:

i) A mouse model for MHC class II deficiency.

In man, the transcription factor RFX5 is required for constitutive and interferon-gamma (IFNg) inducible expression of multiple genes involved in MHC class II (MHC-II) restricted antigen presentation. Mutations within the human RFX5 gene cause a severe immunodeficiency syndrome resulting from the lack of MHC-II expression (MHC-II deficiency). To establish a mouse model for MHC-II deficiency we have used gene targeting to generate mice deficient for the RFX5 gene. The RFX5-/- mouse was generated in collaboration with the laboratory of Professor K. Rajewsky and Dr. I. Forster (Cologne, Germany).

The phenotype of the RFX5-/- mice reproduces all of the major features characteristic of the human disease. MHC-II expression is severely perturbed in all of the cell types that should normally express them. Constitutive MHC-II expression is completely lost on resting B cells, macrophages and the majority of dendritic cells (DCs) in vivo. Nor can peritoneal macrophages be induced to express MHC-II molecules by in vitro stimulation with IFNg. Although a small subpopulation of mature DCs, and B cells stimulated in vitro with LPS and/or IL-4, retain the ability to express MHC-II, they do so at a significantly reduced (10 fold) level. Finally, there is also an essentially complete lack of MHC-II in the thymic cortex, which leads to a severe impairment of positive selection of single positive CD4+ thymocytes, and thus results in a profound peripheral CD4+ T cell deficiency. Accordingly, RFX5-/- mice fail to mount T cell-dependent immune responses.

The RFX5-/- mouse represents a faithful model for human MHC-II deficiency, and will prove to be very useful for the development of gene therapy strategies for this disease. In this context, attempts to restore MHC-II expression by reintroducing an RFX5 transgene with existing and novel lentiviral vectors have been initiated in collaboration with the laboratory of Professor D. Trono (Geneva).

In addition to providing a model for MHC-II deficiency, the RFX5-/- mouse also represents a versatile model system for the development of vectors and approaches designed to deliver and restrict expression of a model transgene (RFX5) to various different cellular compartments in vivo. These include hematopoietic stem cells, B Iymphocytes, macrophages, DCs, thymic epithelium and a wide variety of IFNg inducible cell types such as endothelial cells, epithelial cells, fibroblasts, hepatocytes and glial cells. The RFX5-/- mouse will thus be exploited to develop regulatory systems permitting tissue specific and/or inducible transgene expression. RFX5 will be used as the model transgene. The regulatory systems used to restrict and manipulate RFX5 transgene expression will be based on improved versions of the existing tetracycline controlled expression systems.

The goal of this project is the development and optimisation of regulatory systems designed to achieve tissue specific and tetracycline inducible transgene expression in an in vivo setting, namely in mouse models for a hereditary human immunodeficiency disease. Progress has been made in the following four key aspects of the project.
a) A mouse model for MHC class II deficiency. To develop an in vivo system in which regulated expression of a model transgene can be readily analysed, we have constructed a mouse model for a hereditary immunodeficiency syndrome called MHC class II (MHC-II) deficiency. We have reproduced this disease in mice by using gene targeting to disrupt the murine gene encoding RFX5, an essential transcription factor for MHC-II genes. The RFX5-/- mouse represents a faithful animal model for MHC-II deficiency and is being used for the development of gene therapy strategies for this disease. Pilot experiments aimed at restoring RFX5 expression in these mice with novel lentiviral based vectors have been initiated (see below). Similar experiments with a second model for MHC-II deficiency (CIITA deficient mice) are also in progress.
b) The development of bicistronic lentiviral vectors. We have developed a series of bicistronic lentiviral vectors designed to deliver the MHC-II regulatory genes RFX5, RFXAP, RFXANK and CIITA into cells from MHC-II deficiency patients having defects in the corresponding genes. These vectors are also being used to re-introduce RFX5 or CIITA transgenes into cells from RFX5-/- and CIITA-/- mice (see above). The bicistronic vectors are capable of transducing and complementing the MHC-II deficient cells with high efficiencies. Moreover, they are designed to permit simple and efficient purification of the transduced cells. Finally, the same bicistronic lentiviral vector approach is also being used to deliver the improved tetracycline regulated transactivators we have constructed (see below).
c) Improved tetracycline inducible systems. The RFX5-/- and CIITA-/- mice represent excellent in vivo models of general value for the development of regulatory systems designed to control the expression of typical transgenes (RFX5 and CIITA) in a variety of different cell types. To control transgene expression we intend to use regulatory systems relying on tetracycline controlled transcriptional activators. For this purpose, we have modified existing tetracycline controlled transcriptional activators in order to create "humanised" versions that are more suitable for use in mammalian cells. These humanised versions are significantly more efficient than the original versions, both in transient transfection experiments and in cells transduced stably with lentiviral vectors.
d) DC specific transgene expression. Among the cell types in which it would be useful to restrict transgene expression, dendritic cells (DCs) are of special interest in the context of gene therapy approaches aimed at manipulating the immune response (e.g. vaccination, tumour immunotherapy). We are therefore exploring the possibility of using a novel DC specific promoter, namely promoter I of the CIITA gene. We have confirmed that CIITA promoter I is used specifically in splenic and bone marrow derived DCs in the mouse, and could thus be used to direct DC specific transgene expression. These results have prompted us to replace the constitutive CMV promoter in our lentiviral vectors with promoter I of the CIITA gene. This is planned both for the vectors that express the MHC-II regulatory genes and for those expressing the tetracycline regulated activators. DC specific expression of RFX5 and CIITA will be analysed by transduction of various different cell types derived from the RFX5-/- and CIITA -/- mice. DC specific expression of the tetracycline regulated activators will be analysed by transduction of various different cell types derived from transgenic mice carrying a tet-operator-luciferase reporter gene.

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