Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-13
2001-10-16
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S455000, C536S023100
Reexamination Certificate
active
06303300
ABSTRACT:
The present invention relates to the field of synthetic transfection systems useful in the delivery of gene constructs or DNA fragments to cells, especially to cells in living organisms. More in particular, the invention relates to cationic polymers having broad possibilities to be modified or adapted in order to create a flexible DNA gene delivery system, which can be used in, e.g., gene therapy applications.
Gene therapy is seen as a promising method to correct hereditary defects or to treat life threatening diseases such as cancer and AIDS. In gene therapy, nucleic acid fragments or gene constructs are brought into target cells. These nucleic acid fragments or gene constructs are preferably incorporated in plasmids or other vectors.
If the reconstructed plasmids are applied to an organism per se, this generally leads to low expression of the introduced gene, if any. There are three main reasons for this low expression. First, the plasmids will hardly ever reach the cell population where they are intended to be incorporated, due to degradation and elimination processes. Second, if the plasmids do reach the target cells, they cannot simply pass the cellular membrane, because of the strongly polar nature and the size of the plasmids. Third, if a plasmid does invade a target cell, it normally will be enclosed in an endosome, which will convert into a lysosome. In the lysosome, the plasmid will be degraded so that the incorporated gene cannot be expressed.
For the above reasons, in gene therapy plasmids comprising a desired gene construct are transported to and delivered in the target cells by means of carrier systems.
In recent years, many efforts have been made in the research on potentially suitable transfection systems, both of viral and non-viral origin. These transfection systems should deliver the desired gene to the target cell and cause it to be expressed to a high degree.
Viral vectors are very suitable, because by nature adapted, to introduce plasmids in target cells and to avoid endosome disruption, the degradation of the plasmids in endosomes or the transition of endosomes into lysosomes. However, viral vectors have a number of pronounced disadvantages. Viral vectors are able to effect integration of the introduced gene in the chromosomal DNA of the target cell. The site where this integration is effected cannot (yet) be predicted or controlled, which involves the risk of destroying essential genes or activation of, e.g., oncogenes. In addition, it is at present very difficult to provide for viral vectors on a commercial scale. Moreover, viral vectors generally trigger the immune system of a living organism, which will lead to immuno responses against the transfection system when used in vivo. Finally, viral vectors inherently set limits to the size of the gene construct to be introduced in the target cell.
In order to overcome the intrinsic disadvantages of viral vectors, synthetic transfection systems should offer good perspectives.
In this light, a review article of Kabanov et al. in Bioconjugate Chemistry vol. 6, no. 1 (1995), 7-20 is mentioned. This review article describes in general terms the principle of delivery of genetic material onto cells employing soluble in terpolyelectrolyte complexes (IPEC's) of nucleic acids with linear polycations. It is indicated that various polycations have been used to produce IPEC's. As explicit examples polyvinyl pyrimidinium salts, polypeptides such as polylysine conjugates and lipopolylysines, and spermines are mentioned.
Explicit reference is made to research carried out by the group of E. Wagner, relating to gene delivery by means of plasmid-polylysine complexes (Curiel et al., Adenovirus Enhancement of Transferrin-Polylysine-Mediated Gene Delivery, Proc. Natl. Acad. Sci. 88 (1991) 8850-8854; Plank et al., Gene Transfer into Hepatocytes Using Asialloglycoprotein Receptor Mediated Endocytosis of DNA Complexed with an Artificial Tetra-Antennary Galactose Ligand Bioconj. Chem. 3 (1992) 533-539; Wagner et al., Influenza Virus Hemagglutin HA2 N-Terminal Fusogenic Peptides Augment Gene Transfer by Transferrin-Polylysine-DNA Compleses: Toward a Synthetic Virus-like Gene-Transfer Vehicle, Proc. Natl. Acad. Sci. 89 (1992) 7934-7938; and Curiel et al., Gene Transfer to Respiratory Epithelial Cells via the Receptor Mediated Endocytosis Pathway, Am. J. Respir. Cell Mol. Biol. 6 (1992) 247-252). The plasmid-polylysine complex investigated upon exposition to certain cell lines showed at least some expression of the gene. Further, it was found that the expression efficiency increased considerably due to the binding of transferrin to the plasmid-polylysine complex. Transferrin gives rise to close cell-complex contact; it binds the entire complex to the transferrin receptor of cells. Subsequently, at least part of the entire complex was found to be incorporated in the cell.
However, the transfection efficiency of these polylysine based transfection systems as well as other known synthetic transfection systems is much lower than the efficiency of the known viral vectors.
The aim of the present invention is to provide for an effective and efficient synthetic transfection or blocking system. Such a system should fulfil the following conditions. The synthetic carrier system used must be biocompatible and preferably biodegradable. In order to be able to bind and condense DNA, e.g. in the form of a plasmid, wherein a gene construct is incorporated, the carrier system should possess a positive charge at physiological pH.
It has now been found that such a system can be based on polyphosphazenes which are at least partially substituted with cationic substituents. More in particular, the invention relates to water soluble or water dispersible poly(organo)-phosphazene based transfection systems, wherein organic cationic moieties are attached to the polyphosphazene backbone.
The polyphosphazene polymer which forms the basis of the poly(organo)phospnazene systems of the present invention essentially comprises a backbone of —[—P(R)
2
=N—]
n
— units, wherein (R)
2
represents two groups, which may or may not be the same, coupled to the phosphorous atom. A restricted number of other units may, however, be present.
It is already known from other technical fields that polyphosphazenes are biocompatible and biodegradable. Polyphosphazenes were studied in the art of biomedical and pharmaceutical applications. In this light, reference is made to, e.g., the PhD Thesis of J. Goedemoed titled “Polyphosphazene Drug Delivery Systems for Antitumor Treatment”, University of Leiden (1990); to Crommen et al. Biodegradable Polymers I, Synthesis of Hydrolysis-Sensitive Poly(organo)phosphazenes, Biomaterials 11 (1992), 511-520; to Domb et al. in Polymer Advanced Technology vol. 3, no. 6 (1992) 279-292; to Calicetti et al. in Il Farmaco vol. 49, no. 1, (1994) 69-74; and Andriarov et al. in J. Control Release vol. 27, no. 1 (1983) 69-77. These other technical fields essentially relate to controlled and sustained release systems.
In order to be able to bind to and condense with plasmids, the poly(organo)phosphazene used must—at physiological pH—at least contain 5% cationic radicals or groups coupled to the phosphorous atom in the —[—P(R)
2
=N—]
n
— units. Preferably, at least 40% and more preferably 50% cationic groups are coupled to the phosphorous atoms. In these cases, the polyphosphazene is able to bind DNA electrostatically and condense therewith. Moreover, degradation and elimination of the DNA in the systemic environment is avoided. In addition, it appeared that such polyphosphazene-DNA complexes are taken up in the target cells in a considerably higher amount as compared with the plasmids per se.
Suitable cationic substituents are preferably derived from organic moieties possessing an amino group. Such moieties are positively charged at physiological pH. Examples of these organic moieties are amino C
1-10
alcohols, and amino C
1-10
alkoxy C
1-10
alcohols, as well as their secondary, tertiary and quaternary derivatives
Bout Abraham
Hennink Wilhelmus Everhardus
Introgen B.V.
Morrison & Foerster / LLP
Sandals William
Schwartzman Robert A.
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