Complexes containing nucleic acid which can be taken-up by endoc

Details Complexes containing nucleic acid which can be taken-up by endoc Complexes containing nucleic acid which can be taken-up by endoc

1993-08-05

1999-07-13

Houtteman, Scott W.

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

Organic compounds

Carbohydrates or derivatives

4351721, A61K 4800, C07H 2104

Patent

active

059228598

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The invention relates to the introduction of nucleic acids by endocytosis into higher eukaryotic cells by means of internalizing factors.


BACKGROUND OF THE INVENTION

In recent years, nucleic acids have acquired greater significance as therapeutically active substances, e.g. antisense RNAs and DNAs have proved to be effective agents for selectively inhibiting certain genetic sequences. Their mode of activity enables them to be used as therapeutic agents for blocking the expression of certain genes (such as deregulated oncogenes or viral genes) in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells and perform their inhibiting activity therein (Zamecnik et al., 1986), even though the intracellular concentration thereof is low, partly because of their restricted uptake through the cell membrane owing to the strong negative charge of the nucleic acids.
Another method of selectively inhibiting genes consists in the application of ribozymes. Here again there is the need to guarantee the highest possible concentration of active ribozymes in the cell, for which transportation into the cell is one of the limiting factors.
There is also a need for an efficient system for introducing nucleic acid into living cells in gene therapy. For this, genes are delivered into cells in order to achieve the synthesis of therapeutically active gene products in vivo.
Increasingly, there is a need for methods of treatment in which the therapeutically active DNA (or mRNA) is administered like a drug ("gene therapeutic agent") either once or repeatedly, as required. Examples of genetically caused diseases in which gene therapy constitutes a promising approach are hemophilia, betathalassemia and "Severe Combined Immune Deficiency" (SCID), a syndrome caused by a genetically induced lack of the enzyme adenosine deaminase. Other possible applications are in immune regulation in which a humoral or intracellular immunity is achieved by the administration of functional nucleic acid which codes for a secreted protein antigen or for a non-secreted protein antigen, by means of an inoculation. Other examples of genetic defects in which a nucleic acid coding for the defective gene can be administered, e.g. in a form tailored to the individual requirements, include muscular dystrophy (dystrophin gene), cystic fibrosis ("Cystic fibrosis transmembrane conductance regulator gene") and hypercholesterolemia (LDL receptor gene). Methods of treatment by gene therapy are also of potential significance where hormones, growth factors or proteins with a cytotoxic or immunomodulating activity are to be synthesized in the body.
Numerous solutions have already been proposed for improving the transportation of nucleic acids into living cells, which is one of the limiting factors in the therapeutic use thereof.
One of these possible solutions consists of directly modifying the nucleic acids, e.g. by substituting the charged phosphodiester groups with uncharged groups. Another possible method of direct modification consists in using nucleoside analogues. However, these proposals have various disadvantages, e.g. reduced binding to the target molecule, a poorer inhibitory effect and possible toxicity.
An alternative approach to the direct modification of the oligonucleotides consists in leaving the oligonucleotide per se unchanged and providing it with a group which gives it the desired properties, e.g. with molecules which facilitate transportation into the cells.
There are various known techniques for the genetic transformation of mammalian cells in vitro, but their use in vivo is restricted (they include the introduction of DNA by means of viruses, liposomes, electroporation, microinjection, cell fusion, DEAE-dextran or the calcium phosphate precipitation method).
Attempts have already been made to develop a soluble system which can be used in vivo to convey the DNA into the cells in targeted manner (Wu and Wu, 1987). This system was developed for hepatocytes and is based on the principle of

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