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.
USPTO Backgrounds
USPTO Backgrounds