Conjugates of DNA interacting groups with steroid hormones...

Details Conjugates of DNA interacting groups with steroid hormones... Conjugates of DNA interacting groups with steroid hormones...

2001-03-26

2004-06-22

Badio, Barbara P. (Department: 1616)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

Reexamination Certificate

active

06753424

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides novel compounds that target nucleic acids to the cell nucleus. Further, this invention generally relates to the transformation of cells, particularly mammalian cells, with exogenous DNA or other nucleic acids. More particular, the invention relates to a method for introducing nucleic acids into the nucleus of cells with the help of such compounds. In addition pharmaceutical preparations containing such compounds and the use of such compounds for gene therapy are also in the field to which the invention relates.
BACKGROUND OF THE INVENTION
All references cited herein in short form are outlined in detail in the list of references. The use of gene therapy to treat diseases of both genetic and infectious origin has increasingly become the focus of biomedical research. Accordingly, there have been numerous attempts to develop appropriate delivery systems, based on either recombinant viruses or non-viral vectors.
Several methods have been developed for introducing exogenous DNA molecules into eukaryotic cells for the production of transiently-and stably transfected cells. These methods include physical and chemical systems such as electroporation, microinjection, dextran, liposomes, calcium phosphate or polyethylenimine (PEI) mediated DNA uptake or cell fusion, and microprojectile bombardment. In addition, viral vectors have been used for DNA delivery into cells.
Although physical and chemical methods relatively efficiently overcome the plasma membrane of the cell, it is still unclear how DNA introduced into the cell by these methods penetrate the nuclear envelope. One of the current hypothesis is that the exogenous DNA that survives cytoplasmic degradation is incorporated into the nascent nucleus during cell division. Thus, cytoplasmic degradation as well as the capability of a transfected cell to divide will limit the efficacy of DNA uptake into the nucleus. Furthermore, quiescent, non-dividing cells are rarely transformed by these methods. However, targeting of quiescent cells is of primary importance for somatic gene therapy since a large proportion of somatic cells are non-dividing.
Another limitation of conventional physical and chemical methods is that they cannot provide specificity for particular cell types, i.e. by using a receptor mediated uptake approach. However, this is a highly desired goal if particular cells are to be targeted in tissues or intact organisms, as e.g. in gene therapy applications.
In an attempt to overcome some of the above drawbacks recombinant viral vectors are used for cell transformation. For example, viral systems derived from Adenovirus, Adeno-associated virus, Herpes simplex virus and HIV are being evaluated for targeting of quiescent cells. However, such viral systems pose various problems that are well-known in the art regarding safety in production and application, production costs, efficiency of transfection, duration of expression and amount of DNA that can be packaged, depending on the particular approach used. For example, the use of adenoviral systems is limited by the induction of immune responses to viral antigens with subsequent clearance of transducted cells, thereby strongly diminishing the prospects for long term gene expression. A major safety issue when viral vectors are used is i.e. the generation of replication competent particles during the in vivo packaging of recombinant viruses. This problem is absent if non-viral gene transfer systems are used.
A major advantage of some viral gene delivery systems as compared to conventional physical and chemical methods is the ability of viral vectors to target their DNA-load to the nucleus of the transduced cell, thereby increasing transformation efficiency.
An approach to target DNA into the cell nucleus would be to make use of the cell's own transport mechanisms that specifically guide cytoplasmic molecules through the nuclear pore into the nucleus. In example, certain transcription factors upon activation specifically translocate into the nucleus and, thus, such transcription factors could be used to target molecules to the nucleus. Steroid hormone receptors are an example of transcription factors located in the cytoplasm. They are activated by the binding of steroid hormones and subsequently localize to the nucleus. Use of these receptors for gene delivery systems could therefore accomplish nuclear targeting of the transfected DNA. Via nuclear targeting of the transfected DNA cells could be transfected more efficiently, because breakdown of the nuclear envelope during cell division would not be required to incorporate the transfected DNA. In particular, non-dividing cells could be transformed more efficiently. In addition, cellular targeting the DNA exclusively to cells that express the particular receptor used could be accomplished.
Petros et al. (WO 96/03875) describe a gene delivery system for nucleic acids to cells that comprises a steroid moiety capable of binding to an androgen receptor, wherein the steroid moiety is covalently linked lo a cationic salt, i.e. poly-L-lysine via an ester, an amide or a disulfide bond.
Efficient transformation of cells with this approach is largely dependent on the intracellular stability of the complexes administered. Intracellular conditions, such as i.e. pH, ionic concentrations and the presence of degrading enzymes are some of the limiting factors of intracellular stability of a compound. For example, if ester bonds are present in the complex they could be attacked by intracellular esterases. Further, unfavorable pH and ionic conditions may destabilize ionic linkages, e.g. when polycationic compounds such as poly-L-lysine are used to complex the DNA.
Another factor that may determine the success of a steroid mediated gene delivery approach is the maintenance of a high binding affinity between the steroid moiety and the steroid receptor after the derivatization of the steroid. The size of the DNA interacting moiety, the position of the linkage between the steroid and the DNA interacting moiety, as well as the steric properties of the linking bond itself, may, among others, determine whether steric hindrance of the steroid/steroid receptor interaction will occur after derivatization of the steroid. Further, complexing of the DNA to the DNA interacting moiety should be achieved at specific positions of the DNA molecule in order to avoid inactivation of the genes to be transcribed. For example, complexing the DNA by intercalation may randomly inactivate portions of the DNA. Further, the intercalating positions may even change after initial linkage was achieved, thereby preventing the use of two-step approaches that in a first step will link the DNA interacting moiety to a particular, i.e. non-transcribed DNA region and in a second step will ligate or clamp via bifunctional triple helix formers the functional genes to the complexed DNA region to avoid their inactivation. Also, complexation via cationic moieties interacting with the negatively charged DNA are random and may involve functionally important stretches of the DNA and thus interfere with the transcription of the complexed DNA.
A steroid mediated gene delivery system that combines high intracellular stability and a high binding affinity for the steroid receptor as well as the possibility for specific linkage to the desired DNA molecule has not been reported to date. Thus, there exists a continuous need for such a delivery system which is useful for the introduction of nucleic acids into the nuclei of cells, e.g., for the expression of therapeutical genes. The object of the present invention is therefore to provide such a novel system and new methods for introducing nucleic acids into the nuclei of cells, in particular mammalian cells.
SUMMARY OF THE INVENTION
The present invention provides for a compound comprising a steroid hormone linked to a DNA-interacting molecule. In a preferred embodiment, the steroid hormone is stably linked to the DNA-interacting molecule. This compound is useful for complexing with nucleic acids desired

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