Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-09-01
2004-01-20
Nguyen, Dave T. (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S069100, C435S091300, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06680301
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for introducing molecules in cells by disrupting endosomal and lysosomal membranes using photodynamic treatment, without killing the majority of the cells by the photodynamic treatment. More particularly, this invention includes a method for transferring DNA and/or RNA, such as genes, to cells by photochemically inducing the disruption of endosomes and lysosomes.
BACKGROUND OF THE INVENTION
The majority of molecules do not readily penetrate cell membranes. Methods for introducing molecules into the cytosol of living cells are useful tools for manipulating and studying biological processes. Among the most commonly used methods today are microinjection, red blood cell ghost mediated fusion, liposome fusion, osmotic lysis of pinosomes, scrape loading, electroporation, calcium phosphate, and virus mediated transfection. These techniques are useful for investigations of cells in culture, although in many cases impractical, time consuming, inefficient, or they induce significant cell death. They are thus not optimal for use in biological and medical research or therapeutic applications in which the cells should remain functional.
It is well known that porphyrins and many other photosensitizing compounds induce cytotoxic effects on cells and tissues. These effects are based upon the fact that the photosensitizing compound upon light exposure releases singlet
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which decomposes the membranes of the cells and cell structures, and eventually kills the cells if the destruction is extensive. These effects have been utilized to treat several types of neoplastic diseases. The treatment is named photodynamic therapy (PDT) and is based on injection of a photosensitizing and tumorlocalizing dye followed by exposure of the tumor region to light. The cytotoxic effect is mediated mainly through the formation of singlet oxygen. This reactive intermediate has a very short lifetime in cells (<0.04 &mgr;s). Thus, the primary cytotoxic effect of PDT is executed during light exposure and very close to the sites of formation of
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.
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reacts with and oxidize proteins (histidine, tryptophan, methionine, cysteine, tyrosine), DNA (guanine), unsaturated fatty acids and cholesterol. One of the advantages of PDT is that tissues unexposed to light will not be affected.
There is extensive documentation regarding use of PDT to destroy unwanted cell population, for example neoplastic cells. Several patents relate to photodynamic compounds alone or conjugated with immunoglobulins directed to neoplastic cell receptor determinants making the complex more cell specific. Certain photochemical compounds, such as hematoporphyrin derivates have furthermore an inherent ability to concentrate in malignant cells. These methods and compounds, which are directed to destroy the unwanted cells are described in the Norwegian patent NO 173319, in Norwegian patent applications Nos. 90 0731, 90 2634, 90 1018, 94 1548, 85 1897, 93 4837, 92 4151 and 89 1491.
In PCT/US93/00683 a drug delivery system is described which comprises an anticancer drug and a photoactivatable drug attached to copolymeric carriers. Upon administration this complex enters the cell interior by pinocytosis or phagocytosis and will be located inside the endosomes and lysosomes. In the lysosomes the bond between the antineoplastic compound and the polymer is hydrolyzed, and the former can diffuse passively through the lysosome membrane into cytosol. Thus, this method is limited to small molecular compounds which are able to diffuse across the lysosome membranes. After allowing a time lag for diffusion, a light source of appropriate wavelength is applied to activate the photoactive compound. The combined effects of the anticancer drug and photoactive drug destroy the cell.
Thus, all known use of photoactive compounds is directed to extensively destroy cell structures leading to cell death. A method which releases membrane impermeable molecules into the cytosol after localized rupturing of endosomal/lysosomal membranes is not known.
Desirable molecules for delivery into the cytosol include DNA and/or RNA in the form, for example, of oligonucleotides, oligodeoxynucleotides, ribozymes, antisense molecules, coding sequences, and the like. Once delivered these molecules can be transcribed, expressed, disrupt gene expression, transcription, or translation, or have like effects. For example, gene therapy, i.e. therapeutic transfer of genes to a patient's cells, is a promising method for treating many genetic disorders such as cancer, cystic fibrosis, cardiovascular diseases and many other diseases. Gene therapy includes delivery of a DNA or RNA into the cytosol of a particular cell followed, typically, by an effect in the cytosol or nucleus. However, the DNA or RNA must be delivered to a particular cell or organ and the expression of a therapeutic gene outside the diseased area often can give severe side effects. Many currently existing gene therapy methods lack this desired specificity. Delivery into a cell cytosol or nucleus of DNA and/or RNA for purposes other than gene therapy, for example of antisense or ribozymes, can also be problematic.
SUMMARY OF THE INVENTION
The present invention provides a method to transport molecules, including DNA and/or RNA, into the cytosol of living cells, in culture or in tissues. The method includes exposing the cells to a photoactive compound, or photosensitizer, and the molecule(s) which is (are) to be transported into the cytosol, exposing the cell to light of suitable wavelength to disrupt the endosomal and lysosomal membranes and release the molecules into the cytosol without destroying the functionality of the majority of the cells. Uptake may be facilitated by various carriers. The photosensitizer and the molecule(s) which is (are) to be transported into the cytosol may be conjugated to or applied separately together with suitable carriers, optionally facilitating the uptake of the molecules of interest.
The present invention relates to a method for transporting one or more molecules into the cytosol of a living cell, after which the molecules shall be available in the cytosol and the cell shall maintain its functionability. This is performed by exposing one or more cells to a photoactive compound which is taken up by the cell and will be located in endosomes, lysosomes or other cellular compartments. The photoactive compound is conjugated to or with one or more of a carrier molecule, a targeting immunoglobulin and a molecule to be transported to the cytosol. The cells are then exposed to light of suitable wavelength to activate the photosensitizing compound, such that only the endosomal, lysosomal or other cellular compartment membranes are ruptured, and the molecules are released in the cytosol. Release occurs without the cell losing its functionality by the action of the photoactive compound and possible action of the endosomal/lysosomal content.
The present invention further includes a method for transporting DNA and/or RNA, possibly including one or more genes, via photochemical internalization (PCI) into the cytosol of living cells after which the DNA and/or RNA is available in the cytosol and the cell maintains its functionality. PCI can be used to relocalize DNA and/or RNA oligonucleotides from intracellular granules to the cytosol. The location specificity inherent in the PCI-method can make it possible to specifically deliver DNA and/or RNA to a particular cell type or tissue. For example, PCI can determine nearly exactly to which cell type of an organism the DNA and/or RNA will be delivered and active.
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patent: 5059619 (1991-10-01), Haeger et al.
patent: 5066274 (1991-11-01), Bommer et al.
patent: 5095030 (1992-03-01), Levy et al.
patent: 5095097 (1992-03-01), Hermentin et al.
patent: 5179120 (1993-01-01), Vogel et al.
patent: 5256532 (1993-10-01), Melnicoff et al.
patent: 5264344 (1993-11-01), Sneath
patent: 5328470 (1994-07-01), Nabel et al.
patent: 5374531 (1994-12-01
Berg Kristian
Høgset Anders
Moan Johan
Sandvik Kirsten
Merchant & Gould P.C.
Nguyen Dave T.
Photocure AS
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