Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Patent
1996-12-13
1999-03-30
Low, Christopher S. F.
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
435 52, 514 44, 514 12, 564463, 536 231, 424 145, 424450, 604101, C12N 1587, C12N 1563, C12P 3300
Patent
active
058888217
DESCRIPTION:
BRIEF SUMMARY
This application is a filing under 35 U.S.C. 371 of PCT/DE 95/01879 filed 28 Dec. 1995.
The invention relates to a new cholesterol derivative for liposomal gene transfer. Medicine and genetic engineering are areas of application of the invention.
Cationic liposomes are effective, nonviral transfection reagents for animal cells in vitro (P. Felgner, G. Ringold, Nature 337/1989/, 387-388). The first reagent of this type, DOTMA (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl-ammonium chloride), after being mixed with an equimolar amount of DOPE (dioleyl phosphatidyl ethanolamine), is able to transfect a series of mammalian cells in vitro and in vivo.
The synthesis of DOTMA proceeds over many steps with a relatively low yield. The conventional commercial material, lipofectin, which contains DOTMA and DOPE, furthermore is relatively expensive. Other cationic liposomal reagents with commercially accessible cationic amphiphiles have proven to be relatively toxic with respect to the treated cells (Pinnaduwage et al., Biochim. Biophys. Acta 285/1989/, 33-37).
X. Gao and L. Huang (Biochem. Biophys. Res. Comm. 179/1991/, 280-285) have described the cationic cholesterol derivative 3 .beta.(N-(N',N'-dimethyl-aminoethane)-carbamoyl) cholesterol (DC-Chol). It can be prepared in one step and liposomes with this lipid transfect more efficiently and are less toxic than the lipofectin reagent with respect to the treated cells.
It is an object of the invention to find a new cationic lipid which, while having a transfection ability comparable to that of DC-Chol, is less toxic and, with that, particularly suitable for in vivo applications.
Using DAC-Chol, the new material has the advantage over previously used materials of not being toxic towards highly sensitive cells and of leading to successful transfections in vitro as well as in vivo. The use of the material is demonstrated on glioblastoma cells of the rat, which otherwise can be transfected only with a low effectiveness. Transfection rates are attained, which are up to ten times higher than those attained with the calcium phosphate precipitation technique (CPPT). This finding may well be attributable to a more compact formation of the DNA, a better contact between cells and liposome over the positive charges of the vesicles and to a higher stability with respect to the enzymes breaking down the DNA.
As a result, it can be used advantageously for the direct liposomal gene transfer. Accordingly, immunoliposomes, for example, can also be produced by coupling organ-specific or tissue-specific antibodies with addition of DAC-Chol/DOPE. By a prior incubation of the DNA, which is to be transfected, with nucleoproteins (such as HMG-1), an increased integration and expression of the foreign gene can be attained after encapsulation or association in DAC-Chol/DOPE liposomes. A further increase in the uptake (fusion) of the DAC-Chol liposomes is possible, if fusion proteins or inactivated viruses are reconstituted or associated in the liposomal membrane.
It is furthermore of advantage that these liposomes can be administered without toxicities or immune reactions worth mentioning over automatic or refillable pump systems for a direct in vivo gene transfer (intratumoral or organ-specific). With this method, which can also be employed for other liposomes, a transfection effectiveness is attained, which is much higher than that attained with retroviral or adenoviral in vivo methods. With that, tumor cells, which are in the process of proliferating to different extents at a particular time, can be transfected and killed.
The invention will be described in greater detail in the following by means of examples.
EXAMPLES
cholesterol (DAC-Chol)
The symmetric form of dimethylethylenediamine is reacted by the method of Gao et al. (Biochem. Biophys. Res. Comm. 179; 280) with chloroformyl cholesterol. The oily product obtained is subjected to column chromatography (silica gel 60, with a 9:1 (vol/vol) mixture of trichloromethane and methanol as solvent). The DAC-Chol isolated, after thin-layer ch
REFERENCES:
patent: 4084010 (1978-04-01), Takemoto et al.
patent: 5260290 (1993-11-01), De Luca et al.
patent: 5283185 (1994-02-01), Epand et al.
patent: 5328470 (1994-07-01), Nabel et al.
Pinnaduwage, P., et al; Use of a Quaternary Ammonium Detergent in Liposome Mediated DNA Transfection of Mouse L-cells; Biochimica et Boiphysica Acta, 985 (1989) pp. 33-37.
Gao, Ziang, et al; A Novel Cationic Liposome Reagent for Efficient Transfection of Mammalian Cells; Biochemical and Biophysical Research Communications; vol. 179, No. 1, 1991; pp. 280-285.
Sigma Catalog, 1990, "Biochemicals Organic Compounds", St. Louis, MO, p. 419.
Low Christopher S. F.
Max-Delbruck-Centrum Fur Molekulare Medizin
LandOfFree
Cholesterol derivative for liposomal gene transfer does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Cholesterol derivative for liposomal gene transfer, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cholesterol derivative for liposomal gene transfer will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1213974