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Encapsulation of Plasmid DNA (Lipogenes) and Therapeutic Agents with Nuclear Localization Signal/Fusogenic Peptide Conjugates into Targeted Liposome Complexes

a technology of lipogenes and plasmid dna, which is applied in the field of gene therapy, can solve the problems of difficulty in efficiently delivering polynucleotide to the tumor site, and the routine use of gene therapy for human disease treatment has not yet been perfected, so as to achieve high therapeutic efficacy, improve the entrance effect, and improve the effect of yield

Inactive Publication Date: 2012-07-19
REGULON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]A method is disclosed for encapsulating DNA and negatively charged drugs into liposomes having a different lipid composition between their inner and outer membrane bilayers. The liposomes are able to reach primary tumors and their metastases after intravenous injection to animals and humans. The method includes micelle formation between DNA with a mixture of cationic lipid and peptide molecules at molar ratios to nearly neutralization ratios in 10-90% ethanol; the cationic peptides specify nuclear localization and have a hydrophobic moiety endowed with membrane fusion to improve entrance across the cell membrane of the complex. These peptides insert with their cationic portion directed toward condensed DNA and their hydrophobic chain buried together with the hydrophobic chains of the lipids in the micelle membrane monolayer. The DNA/lipid/peptide micelles are converted into liposomes by mixing w

Problems solved by technology

However, despite numerous preclinical and clinical studies, routine use of gene therapy for the treatment of human disease has not yet been perfected.
One major hurdle preventing successful implementation of these gene therapies is the difficulty of efficiently delivering an effective dose of polynucleotides to the site of the tumor.

Method used

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  • Encapsulation of Plasmid DNA (Lipogenes) and Therapeutic Agents with Nuclear Localization Signal/Fusogenic Peptide Conjugates into Targeted Liposome Complexes
  • Encapsulation of Plasmid DNA (Lipogenes) and Therapeutic Agents with Nuclear Localization Signal/Fusogenic Peptide Conjugates into Targeted Liposome Complexes
  • Encapsulation of Plasmid DNA (Lipogenes) and Therapeutic Agents with Nuclear Localization Signal/Fusogenic Peptide Conjugates into Targeted Liposome Complexes

Examples

Experimental program
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Effect test

example 1

[0172]Plasmid DNA condenses with various agents, as well as various formulations of cationic liposomes. The condensation affects the level of expression of the reporter beta-galactosidase gene after transfection of K562 human erythroleukemia cell cultures. Liposome compositions are shown in the Table below and in FIG. 2. All lipids were from Avanti Polar Lipids (700 Industrial Park Drive, Alabaster, Ala. 35007). The optimal ratio of lipid to DNA was 7 nmoles total lipid / μg DNA. The transfection reagent (10 μg DNA mixed with 70 nmoles total lipid) was transferred to a small culture flask followed by the addition of 10 ml K562 cell culture (about 2 million cells total); mixing of cells with the transfection reagent was at 5-10 min after mixing DNA with liposomes. Cells were assayed for beta-galactosidase activity several times at 1-30 days post-transfection. The transfected cells were maintained in cell culture as normal cell cultures.

[0173]Best results were obtained when the cells us...

example 2

Targeting Genes to Tumors Using Gene Vehicles (Lipogenes)

[0175]As shown in FIG. 3, tumor targeting in SCID (severe combined immunodeficient) mice were implanted subcutaneously, at two sites, with human MCF-7 breast cancer cells. The cells were allowed to develop into large, measurable solid tumors at about 30 days post-inoculation. Mice were injected intraperitoneously with 0.2 mg plasmid pCMVβ DNA (size of the plasmid is ˜4 kb) per animal carrying the bacterial beta-galactosidase reporter gene. Plasmid DNA (200 μg, 2.0 mg / ml, 0.1 ml) was incubated for 5 min with 200 μl neutral liposomes of the composition 40% cholesterol, 20% dioleoylphosphatidylethanolamine(DOPE), 12% palmitoyloleoylphosphatidylcholine (POPC), 10% hydrogenated soy phosphatidylcholine (HSPC), 10% distearoylphosphatidylethanolamine (DSPE), 5% sphingomyelin (SM), and 3% derivatized vesicle-forming lipid M-PEG-DSPE.

[0176]At this stage, weak complexation of plasmid DNA with neutral (zwitterionic) liposomes takes place....

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Abstract

A method is disclosed for encapsulating plasmids, oligonucleotides or negatively-charged drugs into liposomes having a different lipid composition between their inner and outer membrane bilayers and able to reach primary tumors and their metastases after intravenous injection to animals and humans. The formulation method includes complex formation between DNA with cationic lipid molecules and fusogenic / NLS peptide conjugates composed of a hydrophobic chain of about 10-20 amino acids and also containing four or more histidine residues or NLS at their one end. The encapsulated molecules display therapeutic efficacy in eradicating a variety of solid human tumors including but not limited to breast carcinoma and prostate carcinoma. Combination of the plasmids, oligonucleotides or negatively-charged drugs with other anti-neoplastic drugs (the positively-charged cis-platin, doxorubicin) encapsulated into liposomes are of therapeutic value. Also of therapeutic value in cancer eradication are combinations of encapsulated the plasmids, oligonucleotides or negatively-charged drugs with HSV-tk plus encapsulated ganciclovir.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60 / 210,925 filed Jun. 9, 2000. The contents of this application is hereby incorporated by reference into the present disclosure.FIELD OF THE INVENTION[0002]The present invention relates to the field of gene therapy and is specifically directed toward methods for producing peptide-lipid-polynucleotide complexes suitable for delivery of polynucleotides to a subject. The peptide-lipid-polynucleotide complexes so produced are useful in a subject for inhibiting the progression of neoplastic disease.BACKGROUND OF THE INVENTION[0003]Throughout this application various publications, patents and published patent specifications are referenced by author and date or by an identifying patent number. Full bibliographical citations for the publications are provided immediately preceding the claims. The disclosures of these publications, patents and publishe...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61K38/20A61K38/21A61K9/127A61P35/00A61K9/107
CPCA61K9/1075A61K9/1271A61K9/127A61K9/1278C12N15/88A61K9/1277A61P35/00
Inventor BOULIKAS, TENI
Owner REGULON
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