Doctoral thesis

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Nuclear receptors as vehicles for gene transfer


116 p

Thèse de doctorat: Université de Fribourg , 2000

English Human gene therapy can be defined as the directed transfer of exogenous genes or other nucleotide sequences into somatic cells, for the purpose of preventing, correcting or healing various diseases. This novel approach to the therapy of human disorders is based on the realization that many previous medical treatments have usually been directed at the consequences of causative defects or at disease symptoms rather than at the underlying causes. The field of gene therapy has been criticized for promising too much and providing too little during its first 10 years of existence. However, several recent successes achieved in human clinical trials are demonstrating that the concept of gene therapy is finally succeeding. The basic challenge in gene therapy is to develop approaches for delivering genetic material to the appropriate cells of the patient in a way that is specific, efficient and safe. This problem of "drug delivery," where the gene is a drug, is particularly challenging for genes which are large and complex and require targeting to the nuclei of cells. If genes are appropriately delivered they can persist for the life of the cell and potentially lead to a cure. The enabling technology of gene therapy is based on strategies for delivering genes. To do this, special gene delivery vehicles - also called vectors - have been developed. Vectors generally fall into two categories: viral and nonviral. Viral vectors are generally replication defective viruses with part or even all of the viral coding sequence replaced by that of therapeutic genes. Currently, the majority of the vectors for human gene therapy treatments is of viral origin. However, humans have an immune system able to fight off viruses, and attempts to deliver genes via viral vectors have been confronted by these host response. This important drawback of viral systems make synthetic vectors an attractive alternative, especially when readministration of the therapeutic genetic material may be necessary. However, nonviral vectors also have important disadvantages, including their low efficacy and their transient gene expression. In the present study, a novel technique to improve both the efficiency and the specificity of nonviral vectors is described. This new gene transfer technology has been called Steroid Mediated Gene Delivery (SMGD). In particular, the possibility to exploit the shuttling action of nuclear receptors to facilitate the nuclear uptake of DNA has been explored. The SMGD has been modelled with the well characterized glucocorticoid receptor system. The rationale was to decorate transgenes with steroids. To this purpose, special bifunctional steroid derivatives all composed by a steroid molecule covalently linked to DNA interacting compound have been synthesized. The final aim of this work was to test the feasibility of the SMGD using the steroid derivative DR9NP, which has been ultimately selected from the initial collection of synthetic conjugates. This study clearly demonstrates that the transfer of steroid-decorated transgenes is selectively enhanced in presence of the glucocorticoid receptor. This work is the first that shows that nuclear receptors can be exploited as gene transfer vehicles. The SMGD approach can now be extended to other ligands that interact with nuclearly-shuttling intracellular receptors and offers thereby an additional selective advantage to the gene transfer-based treatment of somatic tissues that express specific steroid receptors.
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