Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-10-08
2002-04-23
Nguyen, Dave T. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S455000, C424S093200
Reexamination Certificate
active
06376471
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to improved gene transfer methods and, more particularly, methods that enable highly efficient and widespread delivery of selected nucleic acids, to solid organs such as the heart or liver as well as to other solid cell masses such as a solid tumor.
2. Background
Effective delivery of nucleic acid to cells or tissue with high levels of expression are continued goals of gene transfer technology. As a consequence of the general inability to achieve those goals to date, however, clinical use of gene transfer methods has been limited.
Thus, for example, several delivery schemes have been explored for in vivo myocardial gene transfer, but none has proven capable of modifying a majority of cardiac myocytes in a homogeneous fashion. Techniques involving injection directly into the myocardium are considered of limited use because gene expression does not extend significantly beyond the needle track. R. J. Guzman et al.
Circ Res
1993; 73:1202-1207; A. Kass-Eisler
Proc Natl Acad Sci
1993; 90:11498-11502. In one study, percutaneous intracoronary delivery of 10
10
pfu of adenovirus caused infection in only about one-third of the myocytes in the region served by the target artery. E. Barr et al.
Gene Therapy
1994, 1:51-58.
Other coronary delivery models, either in situ or ex vivo, have produced a very small percentage of infected cells spread throughout the heart. J. Muhlhauser et al.
Gene Therapy
1996; 3:145-153; J. Wang et al.
Transplantation
1996; 61:1726-1729. To date, no in vivo delivery system has been able to infect a majority of cells in an intact heart.
Certain gene delivery procedures also have been quite invasive and hence undesirable. For example, one report describes essentially complete loss of endothelium by mechanical or proteolytic means to enable gene transfer from blood vessels to cells positioned across interposing endothelial layers. See WO 93/00051.
Certain gene transfer applications also have been explored in other organs such as the liver. In particular, ex vivo strategies have included surgical removal of selected liver cells, genetic transfer to the cells in culture and then reimplantion of the transformed cells. See M. Grossman et al.,
Nat Genet
1994, 6:335-341. Such an ex vivo approach, however, suffers from a number of drawbacks including, for example, the required hepatocyte transplantation. M. A. Kay et al.,
Science
1993, 262:117-119; and S. E. Raper et al.,
Cell Transplant
1993, 2:381-400. In vivo strategies for gene transfer to the liver also have been investigated, but have suffered from low delivery efficiencies as well as low specificity to the targeted tissue. N. Ferry et al.,
Proc Natl Acad Sci USA
1991, 88:8377-8391; A. Lieber et al.
Proc Natl Acad Sci USA
1995, 6:6230-6214; A. L. Vahrmeijer et al.,
Reg Cancer Treat
1995, 8:25-31. See also P. Heikkilia et al.,
Gene Ther
1996, 3(1):21-27.
Gene transfer has been generally unsuccessful in additional applications. For example, gene transfer therapies for treatment of cystic fibrosis have largely failed because transduction of insufficient numbers of cells.
It thus would be desirable to have improved methods and systems to effectively deliver nucleic acid to targeted cells and tissue. It would be particularly desirable to have new methods and systems for effective delivery of nucleic acids into solid organs, especially the heart, liver, lung and the like, as well as other solid cell masses such as a solid tumor.
SUMMARY OF THE INVENTION
We have now found methods and compositions that enable effective delivery of nucleic acids to desired cells, including to a solid mass of cells, particularly a solid organ such as a mammalian heart, liver, kidney, skeletal muscle, spleen or prostate, or to malignant cells such as a solid tumor. These methods and compositions enable effective gene transfer and subsequent expression of a desired gene product to a majority of cells throughout a solid cell mass, and/or gene transfer and subsequent expression of a desired gene product to a solid cell mass in a desired percentage of total cells of the mass, including up to nearly 100% of targeted cells of the mass. For example, using methods and compositions of the invention, greater than 90 percent of total cardiac myocytes showed expression of nucleic acid that was perfused for two minutes through an intact rabbit heart.
Methods and compositions of the invention preferably provide enhanced vascular permeability that enables increased nucleic acid delivery to targeted cells. While not being bound by theory, it is believed these methods and compositions of the invention induce transient permeability or interruption of endothelial layers to thereby enhance gene transfer efficiency. This is distinguished from prior approaches that significantly degraded or injured endothelial cell layers in attempts to administer nucleic acid.
Such enhanced permeability can be readily accomplished by one of several alternative approaches, or by a combination of strategies. A preferred approach provides for use of a vasculature permeability agent. As demonstrated in the Examples which follow, use of a suitable permeability agent significantly enhances transfer of administered nucleic acid to targeted cells. A permeability agent suitably may be administered through the vasculature of targeted tissue prior to administration of nucleic acid, and/or the permeability agent and exogenous nucleic acid can be administered simultaneously. Preferably, the vasculature of targeted tissue is pretreated with a permeability agent.
Preferred vasculature permeability agents include serotonin and bradykinin. Other suitable permeability agents will include platelet-activating factor (PAF), prostaglandin E
1
(PGE
1
), histamine, vascular endothelium growth factor (VEGF), zona occludens toxin (ZOT), interleukin-2 and other plasma kinins in addition to bradykinin. Nitric oxide inhibitors, e.g. L-N-monomethyl arginine (L-NMMA) and L-N-nitro-arginine methyl ester (L-NAME), also can provide suitable results, although these agents may be less preferred than others such as serotonin and bradykinin. Other suitable agents can be readily identified, e.g. simply by testing a candidate permeability agent to determine if it enhances uptake of nucleic acid by targeted tissue relative to a control tissue sample that has not been exposed to the candidate permeability agent. A single or a combination of more than one distinct permeability agents may be administered in a particular application. In this regard, a particular application can be optimized by selection of an optimal permeability agent, or optimal “cocktail” of multiple permeability agents. Such optional agent(s) can be readily identified by those skilled in the art by routine procedures, e.g. testing selected permeability agents and combinations thereof in in vivo assays.
Low extracellular calcium ion concentration conditions also can be used to enhance vascular permeability. It has been found that transfer of administered nucleic acid to targeted cells is substantially enhanced under such conditions, which also is demonstrated in the Examples which follow. Low calcium concentration conditions may be readily provided, particularly by perfusing a low calcium ion concentration fluid through the vasculature of the tissue to which nucleic acid is administered. Suitable perfusate calcium ion concentrations may range from about 40 or 50 &mgr;mol/L to about 500 &mgr;mol/L, more preferably from about 50 &mgr;mol/L to about 200 &mgr;mol/L. A perfusate calcium concentration of about 50 &mgr;mol/L is particularly preferred. Calcium ion (e.g. Ca
2+
) concentration also can be lowered through use of a suitable buffer such as a chelating agent, e.g. ethylenebis(oxyethylenenitrilo)tetracetic acid (EGTA), ethylenediaminetetracetic acid (EDTA), or 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA).
Additionally, while a low calcium ion concentration can enhance nucleic acid uptake, it is also importan
Donahue J. Kevin
Lawrence, III John H.
Corless Peter F.
Edwards & Angell LLP
Johns Hopkins University
Nguyen Dave T.
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