NFP37 SOMATIC GENE THERAPY Scientific Abstracts phase B

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FREY, Felix Julius	Inselspital, Abt. für Nephrologie, Dep. für Innere Medizin, Freiburgstrasse 3, CH-3010 Bern tel 031 632 3116; fax 031 632 9734 e-mail: Felix.Frey@insel.ch
Title:	Steroid-mediated gene delivery (SMGD) for therapy of 11beta-hydroxy-steroid dehydrogenase deficiency
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SCIENTIFIC ABSTRACT AT SUBMISSION (1998):

When performing gene transfer in mammalian cells one is confronted with two major barriers: the plasma membrane and the nuclear envelope (see Figure 1). Many efforts in the past have been devoted to the question to know how to overcome the cell membrane. In the previous proposal and in this extension we focus on improving the efficiency of translocation of the transfected DNA from the cytoplasm into the nucleus. For that purpose we take advantage of the natural cytoplasm nucleus ferrying system, represented by steroid receptors. The rationale behind our strategy (named SMGD, Steroid Mediated Gene Delivery) is to tether steroid hormone molecules to the DNA to be transfected. The cognate intracellular steroid receptor is thereby expected to actively increase the nuclear uptake of steroid hormone decorated DNA. In our initial experiments we have modeled the SMGD approach by using glucocorticoid ethidium bromide conjugates. We could prove that hormonedecorated DNA gives enhanced expression in comparison with naked DNA and that this phenomenon is restricted to receptor positive cells. The corresponding results are summarized in an enclosed patent application.

Once the solidity of the principle is proved, we can assume that the SMGD can be utilized in all instances where a specific intracellular receptor makes a tissue or cell population targetable. The goal of the proposed investigation is indeed to verify whether the SMGD approach can be ultimately applied for targeting specific tissues in a systemic gene delivery and for improving the efficacy of relevant therapeutic strategies. Specifically, we shall address the following four questions:

i) Which is the most suitable strategy to link psoralen hormone conjugates to transgenes without impairing transgene function?

ii) Can we assemble conjugates with steroid molecules other than glucocorticoids which maintain their affinity for the cognate receptor, biostability and efficacy in the SMGD assay?

iii) Does the SMGD assisted approach provide higher efficacy and specificity of gene transfer than conventional gene transfer techniques, both in cell culture and in animal models ?

iv) Does the SMGD assisted approach provide an advantage with respect to long term expression of the transgene?

Key words: gene transfer, steroid receptor, beta galactosidase, green fluorescent protein, nude mice, tumor, kidney, nuclear import, 11,B hydroxysteroid dehydrogenase.

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In the aim of improving the efficiency of human gene transfer, numerous attempts to develop appropriate delivery systems based on either viruses or non-viral vectors have been proposed. We have designed an alternative approach, the so-called SMGD, to improve the nuclear uptake of transfected DNA with the help of the natural shuttle for steroid molecules. The rationale is to derivatize steroids and link them to a DNA molecule by covalent, ionic or hydrophobic bond. We anticipate that with an appropriate formulation, the conventionally transfected steroid-DNA complex will be more efficiently and actively translocated into the nucleus via the cognate intracellular receptor shuttle.

For that purpose we synthesized 17 glucocorticosteroid conjugates (requested patents: WO 0011019 A, WO 001 1018 A) consisting of dexamethasone, various chemical spacer (>C4), and various DNAbinding moiety such as psoralene or PNA. The biological stability was established by incubating the conjugates with different biological extracts and confirming their structural integrity by nuclear magnetic resonance spectroscopy. The affinity for the glucocorticoid receptor (GR) was assessed using binding assays. In order to demonstrate translocation of the conjugates from the cytoplasm to the nucleus by GR, a cell line expressing a GR-GFP chimera protein was used. Conjugates with the high affinity for the GR and a suitable spacer showed high translocation efficiency. The expression of the reporter gene attached to the conjugates was studied in CV-1 GR+ and CV-1 GR-cells. An enhanced expression was observed in GR+ cells compared to cells without GR.

In conclusion, the present data indicate that steroid decorated DNA-conjugates can be translocated by the naturally occurring steroid receptor shuttle from the cytoplasm to the nucleus. Since steroid receptors exhibit cell specific expression the SMGD is a new promising strategy for enhancement of gene targeting in vivo.

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