Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Particulate form
Reexamination Certificate
1999-05-06
2001-11-06
McKelvey, Terry (Department: 1636)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Particulate form
C424S493000, C435S320100, C435S455000, C536S023100
Reexamination Certificate
active
06312727
ABSTRACT:
The present invention relates to the delivery of nucleic acid material to target cells in biological systems and to the construction of delivery vehicles for this purpose, especially in connection with gene therapy.
BACKGROUND
The possibility of delivering genes into somatic cells raises many promising new therapeutic opportunities, although the difficulty of efficient delivery to target cells in vivo currently represents a major barrier to progress. Despite the range of techniques available for in vitro transfection of cells, many of these techniques e.g. calcium phosphate precipitation, electro-permeabilisation, etc., cannot be applied in vivo; most animal and clinical studies have relied on the use of liposomal or viral vectors. Cationic liposomes have shown some success in vivo, particularly via non-systemic routes, but they are poorly defined and their net charge is thought to inhibit effective systemic delivery because it promotes binding to plasma proteins and to extracellular matrix. At present viruses provide the most popular vectors for in vivo delivery, particularly with improved DNA packaging techniques. However, their inherent immunogenicity, possibility of fixing complement, poor target-selectivity and difficulty of scale-up production, together with concerns over potential toxicity, seem likely to prevent their widespread acceptance and licensing. There is therefore a clear need for alternative safe and efficient DNA or gene delivery systems, preferably based on fully synthetic carrier vehicles.
A synthetic carrier vehicle or vector suitable for efficient targeted delivery of DNA or other nucleic acid material in vivo must fulfil various biological requirements. Ideally it would be stable in the blood circulation, non-immunogenic and resistant to enzymatic degradation, capable of efficient target-discrimination, and able to penetrate the target cell membrane selectively to gain access to the nucleus, release the nucleic acid and enable efficient transcription within the target cell.
One approach to the development of synthetic vectors or carrier vehicles for delivery of DNA has been proposed based on soluble cationic polymers designed to self-assemble with DNA of expression vectors, it having been shown previously that DNA can be condensed into polyelectrolyte complexes by the addition of polycations, rendering it easier to package. For example, simple mixing of DNA with poly(L)lysine results in formation of discrete polyelectrolyte particles whose size and capacity for spontaneous transfection can be influenced by the molecular weight of the poly(L)lysine used. Specific cell targeting groups, e.g. transferrin and/or membrane-permeabilising groups such as membrane disrupting oligopeptides, can be incorporated into such structures and significantly enhance the transfection rates achieved.
These simple polyelectrolyte DNA complexes are of limited usefulness, however, for systemic administration due to rapid clearance following intravenous (i.v.) injection although the exact reasons for this rapid clearance are not fully understood. However, it does appear that these simple DNA/cationic polymer complexes are subject to destabilisation by serum proteins, especially albumin at physiological concentrations, and may be subject to degradation by serum nucleases, despite their relative stability compared with free DNA.
For successful and versatile in vivo application it is very important that nucleic acid delivery vehicles should be small enough to gain access to target cells. Access to target cells frequently involves extravasation through endothelial layers, but even the hyper-permeable endothelia associated with tumours have a size restriction of about 70 nm. In addition, most forms of triggered membrane penetration act via the endosomal membrane following endocytosis, and endocytic internalisation is usually limited to materials of less than 100 nm diameter. Given the large size of DNA expression vectors in free solution (typical diameter 200 nm) it is clearly necessary for the DNA to be compressed and packaged during self-assembly with cationic polymers if the polyelectrolyte complexes thereby formed are to provide satisfactory DNA carriers and delivery vehicles.
One object of the present invention is to provide improved synthetic polymer-based polyelectrolyte vectors to serve as carrier vehicles for efficient and effective delivery of nucleic acid material, and transfection of target cells, especially in connection with gene therapy or even possibly in connection with development of DNA vaccines.
SUMMARY OF THE INVENTION
Preparation of synthetic polyelectrolyte vectors or nucleic acid carrier vehicles in-accordance with the invention will usually involve forming a complex having a nucleic acid-containing core portion by self-assembly between cationic polymer material and nucleic acid material, especially DNA contained in an expression vector, said core portion being provided, directly or indirectly, with various other functional molecules or molecular entities including molecules of hydrophilic polymer material that provide a coating and steric shield for the core portion, thereby improving stability and biocompatibility of the polyelectrolyte complex. In one technique, this may be achieved by synthesising or modifying a soluble synthetic cationic polymer so as to include or incorporate therein discrete reactive groups prior to arranging a selective self-assembly of such polymer with the nucleic acid material. This self-assembly with the nucleic acid involves an association or binding between molecules of the polycationic component and the polyanionic nucleic acid component. In the complex so formed, the nucleic acid is condensed in the core portion and at least some of said reactive groups on the molecules of the cationic polymer component are presented at the surface thereof. These reactive groups can then be coupled or linked with hydrophilic polymer molecules that associate with the solvent and form in effect an outer shield around the polymer nucleic acid complex, thereby improving stability of the complex and presenting a hydrophilic steric barrier to interactions with cells and molecules which may be encountered in the course of in vivo gene therapy, e.g. while circulating in the plasma after i.v. administration.
In some preferred embodiments the hydrophilic polymer molecules will be multivalent, i.e. will include multiple reactive groups, so that after a first reactive group binds to a reactive group of the cationic polymer and “anchors” the hydrophilic polymer other reactive groups of the latter will bind to other reactive groups of the cationic polymer, thereby cross-linking the coating or outer surface at the same time as providing a steric shielding of the nucleic acid polycation complex.
It will be understood that the term “reactive group” is used herein to denote a group that shows significant chemical reactivity, especially in relation to coupling or linking reactions with complementary reactive groups of other molecules. Also, the terms cationic and anionic denote materials which in aqueous solution at neutral pH have net positive and negative charges respectively.
Thus, as will hereinafter become apparent, the invention provides a nucleic acid carrier vehicle for delivery of nucleic acid material to target cells in biological systems, e.g. in vivo delivery of genes or therapeutic DNA to a patient in carrying out gene therapy or DNA vaccination treatment, said carrier vehicle being in the form of a polyelectrolyte complex comprising a nucleic acid-containing cationic polymer core associated with hydrophilic polymer material that forms an outer stabilising steric shield or coating. The cationic polymer core, which is generally made up of a plurality of polycation molecules, will usually also be linked, directly or indirectly, to other molecular entities or moieties, especially bioactive molecules, that modify the biological and/or physico-chemical characteristics of the complex to improve suitability for use in delivering the nucleic acid material t
Schacht Etienne H
Seymour Leonard C W
Ulbrich Karel
McKelvey Terry
Pillsbury & Winthrop LLP
Sandals William
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