Leland Stanford Junior University

Frank P Diet

Victor J Dzau

Gary H Gibbons

Michael J Mann

Heiko Von Der Leyen

Naked nucleic acids (DNA, RNA, and/or analogs), drugs, and/or other molecules in an extracellular environment enter cells in living intact tissue upon application of pressure to the cells and extracellular environment. Nucleic acids localize to the cell nuclei. Transfection efficiencies greater than 90% are achievable for naked DNA and RNA. A sealed enclosure, defined by an enclosing means and/or tissue, contains the cells and their extracellular environment. The enclosure is pressurized to an incubation pressure on the order of atmospheres. A protective inelastic sheath may be used to prevent distension and trauma in tissue that is part of the enclosure boundary. Suitable enclosures include pressurization chambers and organs such as blood vessels or the heart. Parts of organs, entire organs, and/or entire organisms are pressurized. Suitable target tissue types include blood vessel (in particular vein) tissue, heart, kidney, liver, and bone marrow tissue. Gene therapy applications include ex-vivo treatment of grafts prior to transplantation, and in-vivo treatment of tissue. Useful therapy targets include cell cycle regulatory genes for blocking cell proliferation, and interleukin (IL) and cell adhesion molecule (CAM) genes for reducing immune responses to grafts.

Intracellular delivery of nucleic acids using pressure

The invention presents a system for inducing cells in living intact tissue, in vivo or ex vivo, to accept nucleotides from their extracellular environment and to localize those nucleotides into the cells' nuclei. This system relies on the fact that, when subjected to high pressure, cells take in nucleotides and localize those nucleotides into their nuclei with a transfection rate of greater than 90% in some cases. This invention employs various techniques for placing under high pressure either cells in isolated tissue cultures, or cells in tissues still connected to a living body. Such pressurization techniques include enclosing the tissue in an impermeable and inelastic sheath and injecting fluid into the tissue until a sufficiently high pressure is reached; placing a tissue culture in a dish and placing the dish in a pressurized chamber; mechanical pressurization techniques such as compression of a space containing fluids; occluding the ingress and egress of a vessel or organ to create a watertight compartment within the vessel or organ; and pressurization of an entire organism after delivery of nucleotide to the extracellular environment. This invention also employs various techniques for delivering a solution or suspension containing the nucleotide into the tissue; such techniques include the use of a syringe-like mechanism and a catheter.

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Koln, Germany

Frank P Diet