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Methods for delivering nucleic acid molecules into cells and assessment thereofRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero Ring, Polynucleotide (e.g., Rna, Dna, Etc.)Methods for delivering nucleic acid molecules into cells and assessment thereof description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060194754, Methods for delivering nucleic acid molecules into cells and assessment thereof. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 09/815,979, to de Jong et al., filed on Mar. 22, 2001 and 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 [0002] 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 the nucleic acids therein. BACKGROUND OF THE INVENTION [0003] A number of methods of delivering nucleic acid molecules, particularly plasmid DNA and other small fragments of nucleic acid, into cells have been developed. These methods are not ideal for delivery of larger nucleic acid molecules. Thus, there is a need for methods of delivering nucleic acid molecules of increasing size and complexity, such as artificial chromosomes, into cells. Methods are required for use with in vitro and in vivo procedures such as gene therapy and for production of transgenic animals and plants. Furthermore, there is a need for the ability to rapidly and simply determine and assess the efficiency of delivery of DNA into cells. [0004] Therefore it is an object herein to provide methods for delivering nucleic acid molecules, particularly larger molecules, including artificial chromosomes, into cells. Methods for assessing delivery are also provided. SUMMARY OF THE INVENTION [0005] Methods for delivery of large nucleic acid molecules into cells are provided. The methods, which can be used to deliver nucleic acid molecules of any size, are suitable for delivery of larger nucleic acid molecules, such as natural and artificial chromosomes and fragments thereof, into cells. The methods are designed for in vitro, ex vivo and in vivo delivery of nucleic acid molecules for applications, including, but limited to, delivery of nucleic acid molecules to cells for cell-based protein production, transgenic protein production and gene therapy. Methods of protein production in cells and in transgenic animals and plants, methods of introducing nucleic acid into cells to produce transgenic animals and plants, and methods for ex vivo and in vivo gene therapy are also provided. [0006] Methods provided herein are designed for delivering a large nucleic acid molecule into a cell, but may also be used to deliver smaller molecules. The methods include the steps of exposing the nucleic acid molecule to a first delivery agent, typically an agent that increases contact between the nucleic acid molecule and the cell; and exposing the cell to a second delivery agent, which is generally different from the first agent, and is particularly an agent, such as energy, that enhances permeability of the cell. Selected delivery agents and combinations thereof are those that result in delivery of the nucleic acid the cell to a greater extent than in absence of the agent or in the presence of one of the agent alone. In all of these methods, if the permeability enhancing agent is energy, such as electroporation or sonoporation, the cell is contacted therewith in the absence of the nucleic acid molecule. [0007] Also provided are methods in which the cells are contacted with a lipid agent, particularly a dendrimer, such as SAINT-2.TM. (1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride, also designated 1-methyl-4-(19-cis,cis-hepatritiaconta-9,28-dienyl) pyridinium chloride), simultaneously with or sequentially with application of energy. The nucleic acid, which is optionally, although preferably not treated with a delivery agent, is contacted with the so-treated cell. [0008] The selected delivery methods vary depending on the target cells (cells into which nucleic acid is delivered), the nucleic acid molecules, and the type(s) of delivery agent(s) selected. Exemplary methods for delivery of large nucleic acid molecules into cells provided herein include, but are not limited to, methods involving any of the following: [0009] mixing the nucleic acid molecule with a delivery agent, such as a cationic lipid that neutralizes the charge of the nucleic acid, and contacting the cell with the mixture of nucleic acid and delivery agent; [0010] contacting a cell with the nucleic acid molecule, and then contacting the cell with a delivery agent or contacting a cell with a delivery agent then contacting the cell with the nucleic acid molecule; [0011] contacting a cell in the absence of the nucleic acid molecule with a delivery agent, applying ultrasound or electrical energy to the cell contacted with the delivery agent, and contacting the cell with the nucleic acid molecule upon the conclusion of the application of the energy; [0012] applying ultrasound or electrical energy to a cell, and contacting the cell, upon conclusion of the application of the energy, with a mixture of the nucleic acid molecule and a delivery agent; [0013] applying ultrasound or electrical energy to a cell, contacting the cell with a delivery agent upon conclusion of the application of the energy and contacting the cell previously contacted with the delivery agent with the nucleic acid molecule; [0014] applying ultrasound or electrical energy to a cell and contacting the cell with the nucleic acid molecule upon conclusion of the application of the energy; [0015] contacting a cell in the absence of the nucleic acid molecule with a delivery agent, applying ultrasound or electrical energy to the cell contacted with the delivery agent, and contacting the cell with a mixture of the nucleic acid and a delivery agent upon the conclusion of the application of the energy. [0016] Although combinations of the above methods may be used, it has been found that any application of energy to the cells must be done prior to introduction of the nucleic acid molecule. [0017] The methods provided herein are intended for delivery of large nucleic acid molecules into cells in a variety of environments for a variety of purposes. For example, nucleic acid molecules greater than about 0.5, 0.6. 0.7, 0.8, 0.9, 1, 5, 10, 30, 50 and 100 megabase pairs may be delivered into cells using the methods provided herein. The methods may be used to deliver the large nucleic acid molecules into cells in vitro or in vivo. [0018] In in vivo applications of the delivery methods, such as in in vivo gene therapy, large nucleic acid molecules may be delivered to cells directly in an animal subject, and in particular human subjects. Reagents can be administered locally or systemically (e.g., in the bloodstream) in the subject. For example, local administration of the nucleic acids, and/or delivery agents, may be into areas such as joints, the skin, tissues, tumors and organs. For systemic administration, the nucleic acid molecules may be targeted to cells or tissues of interest. [0019] The delivery methods provided herein may also be used to deliver large nucleic acid molecules to a target cell in vitro which is then introduced into an animal subject, in particular human subjects, such as may be done, for example, in a method of ex vivo gene therapy. Thus, also provided herein are methods of in vivo and ex vivo gene therapy using the methods for delivering large nucleic acid molecules into cells as provided herein. [0020] In particular embodiments of the methods in which a delivery agent is used, the delivery agent is a cationic compound. Cationic compounds include, but are not limited to, a cationic lipid, a cationic polymer, a mixture of cationic lipids, a mixture of cationic polymers, a mixture of a cationic lipid and a cationic polymer and a mixture of a cationic lipid and a neutral lipid, polycationic lipids, non-liposomal forming lipids, activated dendrimers, ethanolic cationic lipids, cationic amphiphiles and pyridinium chloride surfactants. Continue reading about Methods for delivering nucleic acid molecules into cells and assessment thereof... Full patent description for Methods for delivering nucleic acid molecules into cells and assessment thereof Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods for delivering nucleic acid molecules into cells and assessment thereof patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. 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