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Methods for delivering nucleic acid molecules into cells and assessment thereof

a nucleic acid and cell technology, applied in the field of nucleic acid delivery methods into cells, can solve the problems of unsatisfactory methods for delivering larger nucleic acid molecules, and achieve the effects of rapid, simple and accurate detection of delivery, and rapid quantification of differences in delivery efficiency

Inactive Publication Date: 2005-05-26
GLAXO GROUP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes methods for delivering large nucleic acid molecules into cells for various purposes, such as cell-based protein production, transgenic animal and plant production, and gene therapy. The methods involve exposing the nucleic acid molecule to a first delivery agent, typically a cationic lipid, and then contacting the cell with a second delivery agent, such as energy, to enhance permeability of the cell. The methods can be used in in vivo and ex vivo applications, and can also involve the use of a lipid agent, such as a dendrimer, in combination with energy. Overall, the methods provide a reliable and effective way to deliver large nucleic acid molecules into cells for various applications.

Problems solved by technology

These methods are not ideal for delivery of larger nucleic acid molecules.

Method used

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  • Methods for delivering nucleic acid molecules into cells and assessment thereof

Examples

Experimental program
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example 1

Preparation of Artificial Chromosomes

A. GFP Chromosome Contained in A9 Cell Line Plasmids

[0151] Plasmid pIRES-EGFP (see SEQ ID No. 13, plasmid obtained from Clontech, Calif., and is well known, see, e.g., U.S. Pat. Nos. 6,034,228, 6,037,133, 5,985,577, 5,976,849, 5,965,396, 5,976,796, 5,843,884, 5,962,265, 5,965,396; see, also, U.S. Pat. No. 4,937,190). This plasmid contains the internal ribosome entry site (IRES; Jackson (1990) Trends Biochem. 15:477-483; Jang et al. (1988) J. Virol. 62:2636-2643) of the encephalomyocarditis virus (ECMV) between the MCS and the enhanced green fluorescent protein (EGFP) coding region. This permits the gene of interest (cloned into the MCS) and the EGFP gene to be translated from a single bicistronic mRNA transcript. Plasmid pIRES2-EGFP is designed for selection, by flow cytometry and other methods, of transiently transfected mammalian cells that express EGFP and the protein of interest. This vector can also be used to express EGFP alone or to obt...

example 2

Preparation of Cationic Vesicles

[0186] Vesicles were prepared at a lipid concentration of 700 nmol / ml lipid (cationic lipid / DOPE 1:1) as follows. In a glass tube (10 ml) 350 nmol cationic lipid (SAINT-2) was mixed with 350 nmol dioleylphosphoethanolamine (DOPE), both solubilized in an organic solvent (Chloroform, Methanol or Chloroform / Methanol 1:1, v / v). Dioeylphosphatidylethanolamine (DOPE; Avanti Polar Lipids, Alabaster, Ala.) forms inverse hexagonal phases in a membrane and weakens the membrane. Other effectors that may be used are cis-unsaturated phosphaethanolamines, cis-unsaturated fatty acids, cholesterol. Cis-unsaturated phosphatidylcholines are less effective.

[0187] The solvent was evaporated under a stream of nitrogen (15 min / 250 μl solvent at room temperature). The remaining solvent was removed totally by drying the lipid for 15 min in a desiccator under high vacuum from a vacuum pump. To the dried mixture was added 1 ml ultrapure water. This was vortexed vigorously f...

example 3

Preparation of Cationic Vesicles Via Alcoholic Injection

[0188] In a glass tube (10 ml) 350 nmol cationic lipid (SAINT-2) was mixed with 350 nmol DOPE, both solubilized in an organic solvent (chloroform, methanol or chloroform / methanol 1 / 1). The solvent was evaporated under a stream of nitrogen (15 min / 250 μl solvent at room temperature). The remaining solvent was removed totally by drying the lipid for 15 min under high vacuum. This was then reconstituted in 100 μl pure ethanol.

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Abstract

Methods for delivering nucleic acid molecules into cells and methods for measuring nucleic acid delivery into cells and the expression of the nucleic acids are provided. The methods are designed for introduction of large nucleic acid molecules, including artificial chromosomes, into cells, and are practiced in vitro and in vivo.

Description

RELATED APPLICATIONS [0001] This application is a continuation application of copending U.S. application Ser. No. 09 / 815,981, filed Mar. 22, 2001, published as U.S. appln. No. 2003-0003435, to GARY DE JONG and SANDRA LOUISE VANDERBYL, entitled “METHODS FOR DELIVERING NUCLEIC ACID MOLECULES INTO CELLS AND ASSESSMENT THEREOF.” The subject matter thereof is incorporated in its entirety by reference thereto. [0002] This application is related to U.S. application Ser. No. 09 / 815,979, filed Mar. 22, 2001 and published as U.S. appln. No. 2003-0059940, to GARY DE JONG, SANDRA LOUISE VANDERBYL, VOLKER OBERLE AND DIRK HOEKSTRA entitled “METHODS FOR DELIVERING NUCLEIC ACID MOLECULES INTO CELLS AND ASSESSMENT THEREOF.” The subject matter thereof is incorporated in its entirety by reference thereto.FIELD OF THE INVENTION [0003] The present invention relates to methods of delivering nucleic acid molecules into cells and methods for measuring nucleic acid delivery into cells and the expression of ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K41/00C12N15/63C12N15/88
CPCA61K41/0047C12N15/88C12N15/63
Inventor DEJONG, GARYVANDERBYL, SANDRA
Owner GLAXO GROUP LTD
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