Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-04-21
2001-11-20
Nguyen, Dave T. (Department: 1633)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S325000, C435S069100, C424S450000, C424S484000, C424S486000
Reexamination Certificate
active
06319715
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the area of genetic engineering. More particularly, this invention provides a method for enhancing the delivery of nucleic acids into cells using nanoparticles.
BACKGROUND OF THE INVENTION
Gene therapy and DNA vaccination are important alternatives in the treatment and prevention of inherited (e.g., cystic fibrosis), infectious (e.g., AIDS), acquired (e.g., Parkinson's disease) and neoplastic diseases. Infection by viruses, which is nature's predominant means of DNA exchange, is an efficient mechanism for gene transfer. However, clinical applications of viral mediated gene delivery have been hampered by concerns regarding safety, restricted targeting of specific cell types, limited DNA carrying capacity, recombination and high cost. Furthermore, the toxicity and immunogenicity of viral vectors also hamper their routine use in basic research settings. Therefore, non-viral gene delivery systems, especially transfection enhanced by use of chemicals have become popular in both research and clinical settings.
The efficiency of transfection of non-viral gene delivery systems is not as high as the viral-mediated systems. Consequently, a variety of chemicals have been used to increase the efficiency of nucleic acid delivery. However, these have only resulted in moderate increases in DNA delivery to cells Moreover, toxicity considerations limit the concentrations at which the chemicals can be used (Luo et al., 2000,
Nature Biotechnol.,
18: 33-37).
U.S. Pat. No. 5,460,831 to Kossovsky et al., describes a composition of matter comprising mutilayered biodegradable core particles having a diameter less than 1 &mgr;m and having a coating (such as a carbohydrate) that binds to DNA or RNA that is to be delivered to a cell. The core particles may be further coated with targeting agents to direct DNA or RNA to desired cell types.
For transfection of cells in vitro, several parameters have to be considered. The NA uptake across a membrane is not efficient. Even if it were perfectly efficient, only a fraction of the NA molecules in the fluid could reach the cell surface over the transfection period; this fraction is substantially smaller when realistic rates of DNA uptake are considered. While the number of DNA molecules in the medium can be increased by increasing the total concentration of DNA in the fluid, transfection reagents are toxic and effective only at certain vector:NA stochiometries. Thus, it is not feasible to increase the concentration of DNA to increase the efficiency of transfection.
In view of the above, there is an ongoing need for identification and development of novel methods for enhancing the delivery of nucleic acids (NA) into cells that are simple and cost effective.
SUMMARY OF THE INVENTION
The present invention provides a method for enhancing the delivery of nucleic acids (NA) into cells in vitro. The method comprises incubating NA with nanoparticles. The nanoparticle-NA complexes are then allowed to sediment on to the cells. Delivery of the DNA or RNA into cells is accomplished by standard transfection agents or means.
Accordingly an object of the present invention is to provide a method for increasing the delivery of nucleic acids into cells by incubation of the nucleic acid molecules with nanoparticles and using the nanoparticle:NA complexes for delivery of NA to cells using standard methods of delivery.
Another object of the present invention is to provide a method for increasing the transfection efficiency by incubating the DNA:vector with nanoparticles, and allowing the nanoparticles to contact the cells in the presence of standard transfection agents or means.
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Kneuer, et al. International Journal of Pharmaceutics, 2000, vol. 196, pp. 257-261.*
Eck, et al. Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth edition, 1996. McGraw-Hill, New York, pp. 77-101.
Belcheva Nadya
Han Ernest
Luo Dan
Saltzman W. Mark
Cornell Research Foundation Inc.
Hodgson & Russ LLP
Nguyen Dave T.
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