US5922687A
Expired / Lapsed
US
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Intracellular delivery of nucleic acids using pressure
Assignee: Sandia National Laboratories
•
Filing: 1996-11-07
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Granted: 1999-07-13
•
Expires: 2016-11-07
Abstract
Abstract 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.
Inventors
- 👤 Michael J. Mann
- 👤 Frank P. Diet
- 👤 Victor J. Dzau
- 👤 Gary H. Gibbons
- 👤 Heiko Von der Leyen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 04:33
CPC Classifications
C; C12; C12M; C12M3/00; C12M3/04; A; A61; A61M; A61M5/00; A61M5/14; A61M5/142; A61M5/145; A61M5/1452; A61M37/00; A61P; A61P43/00; C12M23/00; C12M23/02; C12M23/14; C12M35/00; C12M35/04; C12M41/00; C12M41/40; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/111; C12N15/113; C12N15/1136; C12N15/87; C12N15/89; A61M25/00; A61M25/10; A61M2025/1043; A61M2025/105; A61M2025/1052; A61M25/1011; C12N2310/00; C12N2310/30; C12N2310/35; C12N2310/351; C12N2310/3517; C12N2320/00; C12N2320/30; C12N2320/32
Claims Analysis
Total Claims: 95
Claim 1
Independent Claim
65 words
A method of delivering a nucleic acid into a cell, said method comprising the steps of: a) placing said nucleic acid in an extracellular environment of said cell; b) defining an enclosure comprising said cell and said extracellular environment; and c) establishing an incubation pressure within said enclosure whereby an establishment of said incubation pressure facilitates an uptake of said nucleic acid by said cell.
Claim 2
Independent Claim
65 words
A method of delivering a nucleic acid into a cell, said method comprising the steps of: a) placing said nucleic acid in an extracellular environment of said cell; b) defining an enclosure comprising said cell and said extracellular environment; and c) establishing an incubation pressure within said enclosure whereby an establishment of said incubation pressure facilitates an uptake of said nucleic acid by said cell.
Claim 3
Dependent Claim
18 words
The method of claim 1 wherein a boundary of said enclosure is defined substantially by an enclosing means.
Claim 4
Dependent Claim
13 words
The method of claim 2 wherein said enclosing means comprises a pressurization chamber.
Claim 5
Dependent Claim
18 words
The method of claim 1 wherein part of a boundary of said enclosure is defined by a tissue.
Claim 6
Dependent Claim
28 words
The method of claim 4 further comprising the step of establishing a protective means adapted to be placed around said tissue, for preventing a trauma in said tissue.
Claim 7
Dependent Claim
56 words
The method of claim 4 further comprising the steps of: a) establishing a first occlusion in a first organ conduit in communication with an organ; and b) establishing a second occlusion in a second organ conduit in communication with said organ; wherein said substantial part of said boundary is defined by a tissue of said organ.
Claim 8
Dependent Claim
22 words
The method of claim 1 comprising the step of establishing said incubation pressure within an entire organ, said organ comprising said cell.
Claim 9
Dependent Claim
11 words
The method of claim 7 wherein said organ is a heart.
Claim 10
Dependent Claim
21 words
The method of claim 1 comprising the step of establishing said incubation pressure around an organism, said organism comprising said cell.
Claim 11
Dependent Claim
19 words
The method of claim 9 comprising the step of perfusing said nucleotide into a target region of said organism.
Claim 12
Dependent Claim
14 words
The method of claim 1 comprising maintaining said incubation pressure at a predetermined level.
Claim 13
Dependent Claim
19 words
The method of claim 1 wherein said incubation pressure is between 0 atm and 2 atm above atmospheric pressure.
Claim 14
Dependent Claim
21 words
The method of claim 1 wherein said incubation pressure is between 300 mm Hg and 1500 mm Hg above atmospheric pressure.
Claim 15
Dependent Claim
15 words
The method of claim 1 wherein said incubation pressure is higher than 100 mm Hg.
Claim 16
Dependent Claim
15 words
The method of claim 1 comprising maintaining said incubation pressure for a predetermined incubation period.
Claim 17
Dependent Claim
18 words
The method of claim 1 comprising maintaining said incubation pressure for an incubation period longer than 1 minute.
Claim 18
Dependent Claim
18 words
The method of claim 1 comprising maintaining said incubation pressure for an incubation period longer than 1 hour.
Claim 19
Dependent Claim
28 words
The method of claim 1 wherein: a) said cell is a mammalian cell in a tissue; and b) said method comprises establishing said incubation pressure around said tissue.
Claim 20
Dependent Claim
26 words
The method of claim 18 wherein said tissue comprises a part of an organ selected from the group consisting of the liver, kidneys, and bone marrow.
Claim 21
Dependent Claim
31 words
The method of claim 18 wherein said tissue comprises a part of an organ selected from the group consisting of bones, muscles, connective tissue, gastrointestinal organs, endocrine organs, and exocrine organs.
Claim 22
Dependent Claim
14 words
The method of claim 18 wherein said tissue comprises a part of a heart.
Claim 23
Dependent Claim
15 words
The method of claim 18 wherein said tissue comprises a part of a blood vessel.
Claim 24
Dependent Claim
14 words
The method of claim 22 wherein said tissue comprises a part of a vein.
Claim 25
Dependent Claim
11 words
The method of claim 1 wherein said nucleic acid is naked.
Claim 26
Dependent Claim
14 words
The method of claim 1 wherein said nucleic acid is attached to a liposome.
Claim 27
Dependent Claim
15 words
The method of claim 1 wherein said nucleic acid is attached to a viral vector.
Claim 28
Dependent Claim
15 words
The method of claim 1 wherein said nucleic acid is attached to a virus-liposome complex.
Claim 29
Dependent Claim
18 words
The method of claim 27 wherein said nucleic acid is attached to a hemagglutinating virus of Japan-liposome complex.
Claim 30
Dependent Claim
16 words
The method of claim 1 wherein said nucleic acid is attached to a molecule of interest.
Claim 31
Dependent Claim
13 words
The method of claim 29 wherein said molecule of interest comprises a protein.
Claim 32
Dependent Claim
13 words
The method of claim 1 wherein said nucleic acid comprises a deoxyribonucleic acid.
Claim 33
Dependent Claim
13 words
The method of claim 1 wherein said nucleic acid comprises a ribonucleic acid.
Claim 34
Dependent Claim
47 words
The method of claim 1 wherein: a) said nucleic acid comprises a decoy double stranded deoxyribonucleic acid having a binding site; and b) a cellular factor present in said cell is adapted to bind to said binding site; whereby said cellular factor binds to said binding site.
Claim 35
Dependent Claim
12 words
The method of claim 1 wherein said nucleic acid comprises a plasmid.
Claim 36
Dependent Claim
19 words
The method of claim 1 wherein said nucleic acid comprises a regulatory element operatively linked to a coding portion.
Claim 37
Dependent Claim
19 words
The method of claim 35 wherein said regulatory element is selected from the group consisting of promoters and enhancers.
Claim 38
Dependent Claim
19 words
The method of claim 1 wherein said nucleic acid encodes a protein adapted to be secreted by said cell.
Claim 39
Dependent Claim
22 words
The method of claim 1 comprising the step of establishing in vitro said incubation pressure around a cell culture comprising said cell.
Claim 40
Independent Claim
77 words
A system for delivering a nucleic acid into a cell, said system comprising: a) an enclosing means for defining at least part of a boundary of an enclosure, said enclosure containing said cell and an extracellular enviroment of said cell, said extracellular enviroment containing said nucleic acid; and b) a pressurization means for establishing an incubation pressure within said enclosure; whereby an establishment of said incubation pressure facilitates an uptake of said nucleic acid by said cell.
Claim 41
Independent Claim
77 words
A system for delivering a nucleic acid into a cell, said system comprising: a) an enclosing means for defining at least part of a boundary of an enclosure, said enclosure containing said cell and an extracellular enviroment of said cell, said extracellular enviroment containing said nucleic acid; and b) a pressurization means for establishing an incubation pressure within said enclosure; whereby an establishment of said incubation pressure facilitates an uptake of said nucleic acid by said cell.
Claim 42
Dependent Claim
19 words
The system of claim 39 further comprising a delivery means for delivering said nucleic acid to said extracellular environment.
Claim 43
Dependent Claim
13 words
The system of claim 39 wherein said enclosing means comprises an impermeable sheath.
Claim 44
Dependent Claim
20 words
The system of claim 39 wherein said enclosing means comprises an occlusion means for occluding a passage in a tissue.
Claim 45
Dependent Claim
13 words
The system of claim 39 wherein said enclosing means comprises a pressurization chamber.
Claim 46
Dependent Claim
15 words
The system of claim 39 wherein said boundary is defined substantially by said enclosing means.
Claim 47
Dependent Claim
15 words
The system of claim 39 wherein part of said boundary is defined by a tissue.
Claim 48
Dependent Claim
24 words
The system of claim 45 further comprising a protective means adapted to be placed around said tissue, for preventing a trauma in said tissue.
Claim 49
Dependent Claim
13 words
The system of claim 46 wherein said protective means comprises an inelastic sheath.
Claim 50
Independent Claim
88 words
A method of delivering a molecule into a cell, said method comprising the steps of: a) placing said molecule in an extracellular environment of said cell comprising a liquid medium; and b) non-surgical establishing enclosure around said cell and said liquid medium and establishing an incubation pressure around said cell and said extracellular environment; wherein said molecule is selected from the group consisting of a drug, sugar, fatty acid and protein and whereby an establishment of said incubation pressure facilitates an uptake of said molecule by said cell.
Claim 51
Independent Claim
88 words
A method of delivering a molecule into a cell, said method comprising the steps of: a) placing said molecule in an extracellular environment of said cell comprising a liquid medium; and b) non-surgical establishing enclosure around said cell and said liquid medium and establishing an incubation pressure around said cell and said extracellular environment; wherein said molecule is selected from the group consisting of a drug, sugar, fatty acid and protein and whereby an establishment of said incubation pressure facilitates an uptake of said molecule by said cell.
Claim 52
Dependent Claim
11 words
The method of claim 48 wherein said molecule comprises a sugar.
Claim 53
Dependent Claim
12 words
The method of claim 48 wherein said molecule comprises a fatty acid.
Claim 54
Dependent Claim
11 words
The method of claim 48 wherein said molecule comprises a protein.
Claim 55
Dependent Claim
11 words
The method of claim 48 wherein said molecule comprises a drug.
Claim 56
Dependent Claim
13 words
The method of claim 48, wherein said cell is in a living mammal.
Claim 57
Dependent Claim
18 words
The method of claim 48, wherein part of a boundary of said enclosure is defined by a tissue.
Claim 58
Dependent Claim
55 words
The method of claim 54 further comprising the steps of: a) establishing a first occlusion on a first organ conduit in communication with an organ; and b) establishing a second occlusion in a second organ conduit in communication with said organ; wherein said substantial part of said boundary is defined by tissue of said organ.
Claim 59
Dependent Claim
11 words
The method of claim 55 wherein said organ is a heart.
Claim 60
Dependent Claim
20 words
The method of claim 48 comprising the step of establishing said incubation pressure around an organism that comprises said cell.
Claim 61
Dependent Claim
19 words
The method of claim 57 comprising the step of perfusing said molecule into a target region of said organism.
Claim 62
Dependent Claim
15 words
The method of claim 48 comprising maintaining said incubation pressure for a predetermined incubation period.
Claim 63
Dependent Claim
18 words
The method of claim 59 comprising maintaining said incubation pressure for an incubation period longer than 1 minute.
Claim 64
Dependent Claim
18 words
The method of claim 60 comprising maintaining said incubation pressure for an incubation period longer than 1 hour.
Claim 65
Dependent Claim
36 words
The method of claim 48 wherein said cell is in a tissue that comprises a part of an organ selected from the group consisting of bones, muscles, connective tissue, gastrointestinal organs, endocrine organs, and exocrine organs.
Claim 66
Dependent Claim
31 words
The method of claim 48 wherein said cell is in a tissue that comprises a part of an organ selected from the group consisting of the liver, kidneys, and bone marrow.
Claim 67
Dependent Claim
14 words
The method of claim 48 wherein said tissue comprises a part of a heart.
Claim 68
Dependent Claim
15 words
The method of claim 48 wherein said tissue comprises a part of a blood vessel.
Claim 69
Dependent Claim
14 words
The method of claim 65 wherein said tissue comprises a part of a vein.
Claim 70
Dependent Claim
13 words
The method of claim 48 wherein said molecule is attached to a liposome.
Claim 71
Dependent Claim
18 words
The method of claim 48 wherein said uptake reduces an immune response of a patient to said cell.
Claim 72
Dependent Claim
21 words
The method of claim 48 wherein said cell is an organ and said incubation pressure is established within the entire organ.
Claim 73
Independent Claim
91 words
A method of delivering a molecule into a cell, said method comprising the steps of: a) placing said molecule in an extracellular environment of said cell comprising a liquid medium; and b) establishing an enclosure around said cell and said liquid medium and establishing an incubation pressure around said cell and said extracellular environment using external pressurizing means; wherein said molecule is selected from the group consisting of a drug, sugar, fatty acid and protein and whereby an establishment of said incubation pressure facilitates uptake of said molecule by said cell.
Claim 74
Independent Claim
91 words
A method of delivering a molecule into a cell, said method comprising the steps of: a) placing said molecule in an extracellular environment of said cell comprising a liquid medium; and b) establishing an enclosure around said cell and said liquid medium and establishing an incubation pressure around said cell and said extracellular environment using external pressurizing means; wherein said molecule is selected from the group consisting of a drug, sugar, fatty acid and protein and whereby an establishment of said incubation pressure facilitates uptake of said molecule by said cell.
Claim 75
Dependent Claim
18 words
The method of claim 70 wherein a boundary of said enclosure is defined substantially by an enclosing means.
Claim 76
Dependent Claim
13 words
The method of claim 71 wherein said enclosing means comprises a pressurization chamber.
Claim 77
Dependent Claim
28 words
The method of claim 70 further comprising the step of establishing a protective means adapted to be placed around said tissue for preventing a trauma in said tissue.
Claim 78
Dependent Claim
15 words
The method of claim 70, further comprising maintaining said incubation pressure at a predetermined level.
Claim 79
Dependent Claim
19 words
The method of claim 74 wherein said incubation pressure is between 0 atm and 2 atm above atmospheric pressure.
Claim 80
Dependent Claim
21 words
The method of claim 74 wherein said incubation pressure is between 300 mm Hg and 1500 mm Hg above atmospheric pressure.
Claim 81
Dependent Claim
15 words
The method of claim 74 wherein said incubation pressure is higher than 100 mm Hg.
Claim 82
Dependent Claim
20 words
The method of claim 70, wherein said incubation pressure is established in vitro around a cell culture comprising said cell.
Claim 83
Dependent Claim
13 words
The method of claim 70, wherein said external pressurizing means comprises a sheath.
Claim 84
Dependent Claim
21 words
The method of claim 70, wherein said external pressurizing means does not cause continuous flow through the vasculature supplying said cell.
Claim 85
Dependent Claim
13 words
The method of claim 80, wherein said cell is in a living mammal.
Claim 86
Dependent Claim
21 words
The method of claim 80, further comprising the step of establishing said incubation pressure within an organ that comprises said cell.
Claim 87
Dependent Claim
13 words
The method of claim 80, wherein said method is carried out ex vivo.
Claim 88
Dependent Claim
36 words
The method of claim 80, wherein: a) said cell is a mammalian cell in a tissue; and b) said method further comprises establishing said incubation pressure by applying pressure to the outer surface of said tissue.
Claim 89
Dependent Claim
18 words
The method of claim 70, wherein said molecule is delivered to the cavity of a hollow internal organ.
Claim 90
Independent Claim
90 words
A system for delivering a molecule into a cell, said system comprising: a) enclosing means for non-surgically defining at least part of a boundary of an enclosure, said enclosure containing said cell and an extracelllular enviroment of said cell that contains said molecule; and b) a pressurization means for establishing an incubation pressure within said enclosure; wherein said molecule is selected from the group consisting of a drug, sugar, fatty acid and protein and whereby an establishment of said incubation pressure facilitates the uptake of said molecule by said cell.
Claim 91
Independent Claim
90 words
A system for delivering a molecule into a cell, said system comprising: a) enclosing means for non-surgically defining at least part of a boundary of an enclosure, said enclosure containing said cell and an extracelllular enviroment of said cell that contains said molecule; and b) a pressurization means for establishing an incubation pressure within said enclosure; wherein said molecule is selected from the group consisting of a drug, sugar, fatty acid and protein and whereby an establishment of said incubation pressure facilitates the uptake of said molecule by said cell.
Claim 92
Dependent Claim
18 words
The system of claim 86, further comprising a delivery means for delivering said molecule to said extracellular environment.
Claim 93
Dependent Claim
13 words
The system of claim 86, wherein said enclosing means comprises a pressurization chamber.
Claim 94
Dependent Claim
15 words
The system of claim 86, wherein said boundary is defined substantially by said enclosing means.
Claim 95
Dependent Claim
17 words
The system of claim 86, wherein said enclosing means defines the entire boundary of said sealed enclosure.
References Cited (Backward Citations)
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