Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Isomerase
Reexamination Certificate
1999-09-13
2002-05-14
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Isomerase
C435S183000, C530S300000, C530S350000, C536S023700
Reexamination Certificate
active
06387684
ABSTRACT:
BACKGROUND OF THE INVENTION
Nucleic acid delivery into cells, including the cell nucleus, can be accomplished by viral and non-viral means. For example, retroviruses and adeno-associated viruses are efficient delivery vehicles often used in gene therapy protocols, even though safety concerns over the use of viruses in humans remain. These concerns have prodded the development of non-viral delivery means, such as the use of liposomes to encapsulate nucleic acids. Unfortunately, non-viral delivery vehicles are generally inefficient when compared to viral vectors, due in part to cellular compartmentalization and degradation of the nucleic acid taken up by a cell.
SUMMARY OF THE INVENTION
The invention is based on the discovery of new DNA binding domains within human topoisomerase I (Topo I). These new DNA binding domains can be fused to a ligand domain which binds to a molecule on an external surface of a cell, thereby providing an efficient means of delivering DNA to the cell surface and potentially increasing DNA uptake into the cell. An example of such a ligand domain is a receptor binding domain of a Pseudomonas exotoxin A, which specifically binds to the LDL/&agr;
2
-microglobulin cell receptor, a prolific cell surface molecule, especially on hepatocytes.
Accordingly, the invention features a fusion polypeptide having (1) a ligand domain that specifically binds to a molecule on an external surface of a cell, and (2) a DNA binding domain of a Topo I. The fusion polypeptide can further include a membrane translocation domain, e.g., a membrane translocation domain of a Pseudomonas exotoxin A to facilitate delivery of the DNA into the target cell. A membrane translocation domain is an animo acid sequence within a polypeptide which facilitates the translocation of the polypeptide from a non-cytosolic compartment (e.g., endosomes) to the cytosol of a cell.
An example of a membrane translocation domain is amino acids 253-364 of
P. aeruginosa
exotoxin A:
lagnpakhdldikptvishrlhfpeggslaaltahqachlpletf trhrqprgweqleqcgypvqrlvalylaarlswnqvdqvirnala spgsggdlgeaireqpeqarla (SEQ ID NO:6).
Examples of Topo I DNA binding domains include the following human Topo I amino acid sequences:
msgdhlhndsqieadfrlndshkhkdkhkdrehrhkehkkekdre kskhsnsehkdsekkhkekektkhkdgssekhkdkhkdrdkekrk eekvrasgdakikkekengfssppqikdepeddgyfvppkedikp lkrprdeddvdykpkkiktedtkkekkrkleeeedgklkkpknkd kdkkvpepdnkkkkpkkeeeqkwkwweeerypegikwkflehkgp vfappyeplpenvkfyydgkvmklspkaeevatffakmldheytt keifrknffkdwrkemtneekniitnlskc (SEQ ID NO:1),
nsehkdsekkhkekektkhkdgssekhkdkhkdrdkekrkeekvr asgdakikkekengfssppqikdepeddgyfvppkedikplkrpr deddvdykpkkiktedtkkekkrkleeeedgklkkpknkdkdkkv pepdnkkkkpkkeee (SEQ ID NO:2),
keifrknffkdwrkemtneekniitnlskcdftqmsqyfkaqtea rkqmskeeklkikeenekllkeygfcimdnhkerianfkieppgl frgrgnhpkmgmlkr (SEQ ID NO:3), and
gsikyimlnpssrikgekdwqkyetarrlkkcvdkirnqyredwk skemkvrqravalyfidklalragnekeegetadtvgccslrveh inlhpeldgqeyvvefdflgkdsiryynkvpvekrvfknlqlfme nkqpeddlfdrlntgilnkhlqdlmegltakvfrtynasi (SEQ ID NO:4).
SEQ ID NOs:2-4 represent amino acids 51-200, 271-375, and 422-596 of human Topo I, respectively. SEQ ID NO:1 represents a longer sequence (amino acids 1-300) encompassing one of the identified DNA binding domains described above (amino acids 51-200; SEQ ID NO:2).
A ligand domain suitable for use in the fusion polypeptide of the invention is a receptor binding domain of
P. aeruginosa
exotoxin A (PE), such as amino acids 1-252 of PE:
mhliphwiplvaslgllaggssasaaeeafdlwnecakacvldlk dgvrssrmsvdpaiadtngqgvlhysmvleggndalklaidnals itsdgltirleggvepnkpvrysytrqargswslnwlvpighekp snikvfihelnagnqlshmspiytiemgdellaklardatffvra hesnemqptlaishagvsvvmaqtqprrekrwsewasgkvlclld pldgvynylaqqrcnlddtwegkiyrv (SEQ ID NO:5).
The invention also includes a nucleic acid encoding one of the fusion polypeptides described above. In addition, the invention includes a method of producing an above-described fusion polypeptide by introducing a nucleic acid of the invention into a cell and expressing the pQlypeptide in the cell. The nucleic acid can be introduced into the cell by any known method, including transfection or viral delivery.
As used herein, a DNA binding domain is an amino acid sequence which binds DNA of any sequence or topology (e.g., binds to both supercoiled and relaxed circular DNA). All domains can be at least 10 amino acids (e.g., at least 20, 50, 100, 150, or 200 amino acids) in length. They can also be less than 1000 amino acids (e.g., less than 900, 750, 600, or 400 amino acids) in length.
A fusion polypeptide of the invention is useful for delivering any nucleic acid (e.g., a DNA vector), regardless of sequence or topology, into a cell, e.g., for integration and/or expression into a cell genome. In general, the various elements or domains of the polypeptide can in any order from the N-terminus to the C-terminus within the polypeptide, as long as the domains remain functional.
Other features or advantages of the present invention will be apparent from the detailed description, and also from the claims.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to fusion polypeptides containing a DNA binding domain of a human Topo I. Since the DNA binding domain binds DNA of different sequences or topology to a similar degree, the fusion polypeptides are useful for a number of laboratory techniques in which binding of all forms of DNA is beneficial. For example, a fusion polypeptide that contains a DNA binding domain described herein and a ligand for a cell surface receptor can be used to target any DNA molecule to the cell. The receptor binding domain of a
P. aeruginosa
exotoxin A is a suitable ligand for this purpose. Addition of a cellular translocation domain (e.g., the translocation domain of a
P. aeruginosa
exotoxin A) can also be included to further enhance the ability of the fusion polypeptide to deliver DNA into cells.
REFERENCES:
patent: 5668255 (1997-09-01), Murphy
patent: WO 94/04696 (1994-03-01), None
Hwang et al. Development of a DNA delivery system using Pseudomonas exotoxin A and a DNA binding region of human DNA topoisomerase I, Cancer Gene Therapy 5 (6): Supp; O27-Abstract, Dec. 1998.*
Redinbo et al. Crystal Structures of Human Topoisomerase I in Covalent and Noncovalent Complexes with DNA, Science 279: 1504-1513, Mar. 1998.*
Allured et al., “Structure of Exotoxin a of Pseudomonas Aeruginosa at 3.0-Angstrom Resolution”, Proc. Natl. Acad. Sci. USA 83:1320-1324, 1986.
Alsner et al., “Identification of an N-terminal Domain of Eukaryotic DNA Topoisomerase I Dispensable for Catalytic Activity but Essential for in Vivo Function”, Journal Biological Chemistry 267:12408-12411, 1992.
Chow et al., “Identification of the Carboxyl-terminal Amino Acids Important for the ADP-Ribosylation Activity of Pseudomonas Exotoxin A”, The Journal of Biological Chemistry 264:18818-18823, 1989.
Fominaya et al., “Target Cell-specific DNA Transfer Mediated by a Chimeric Multidomain Protein”, The Journal of Biological Chemistry 271:10560-10568, 1996.
Hwang et al., “Functional Domains of Pseudomonas Exotoxin Identified by Deletion Analysis of the Gene Expressed inE. Coli”, Cell 48:129-136, 1987.
Hwang et al., “Structure and Function Relationship of Pseudomonas Exotoxin A”, The Journal of Biological Chemistry 264:2379-2384, 1989.
Chen Tzong-Yueh
Hui Cho-Fat
Hwang Jaulang
Academia Sinica
Hutson Richard
Prouty Rebecca E.
LandOfFree
Topoisomerase 1-mediated DNA delivery does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Topoisomerase 1-mediated DNA delivery, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Topoisomerase 1-mediated DNA delivery will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2891607