Fusion protein for use as vector

Details Fusion protein for use as vector Fusion protein for use as vector

2002-05-13

2004-12-28

Wax, Robert A. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

25 or more amino acid residues in defined sequence

C530S300000

Reexamination Certificate

active

06835810

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a fusion protein referred as a novel vector for delivering molecules into cells.
2. Description of the Prior Art
The transfer of genetic material into cells in mammals is of increasing therapeutic and commercial importance. For instance, gene therapy procedures are used to correct acquired and inherited genetic defects, cancer, and viral infection. The ability to express artificial genes in humans facilitates the prevention and/or cure of many major human diseases, including many diseases that are not amenable to treatment by other therapies. However, biological membranes are natural barriers central to compartmentalization in living systems. Therefore, the polypeptides and oligonucleotides are generally considered to be of limited therapeutic value. Many studies have been conducted to overcome the problem of delivering such polypeptides and oligonucleotides.
Most of the initial work focused on the use of retroviral vectors to transform these cells. However, numerous difficulties with retroviruses have been reported. For example, it is hard to infect certain cell types. Retroviruses typically enter cells via receptors and if such receptors are not present in the cell, or are not present in large numbers, the infection is not possible or efficient.
Many researchers developed liposome systems for delivering the polypeptides and oligonucleotides into the cells. Liposomes are small membrane-enclosed spheres that have been formed with the appropriate DNA entrapped within it. However, this system also has inherent problems. It is difficult to control the size of the liposome and hence the uniformity of delivery to individual cells. Additionally, it is difficult to prevent leakage of the contents of the liposome and as with other techniques, there is difficulty in directing cell-type specificity.
Recently, several small regions of proteins called protein transduction domains (PTDs) have been developed to transport molecules into cells (Fischer et al, Bioconjugate Chem., Vol. 12, No. 6, 2001). Such PTD can translocate the cell membrane freely in a way that is receptor-, or transporter-independent, non-saturable, and consumes no energy. The PTD can get across the barrier of the cell membrane within less than one hour. The fundamental requirements for the creation, isolation and utilization of TAT-fusion proteins to affect mammalian cells were described in Becker-Hapak et al. Methods 24, 247-256, 2001. In Steven et al., a series of synthetic PTDs that strengthen the &agr;-helical content and optimize the placement of arginine residues were synthesized. Several PTD peptides possessed significantly enhanced protein transduction potential compared with TAT (Steven et al., Cancer Research 61, 474-477, Jan. 15, 2001). Furthermore, U.S. Pat. No. 6,090,619 described the preparation of a novel non-viral vector, which can bind to desired DNA to form a combination useful to transfect diseased mitochondria of human or animal cells. U.S. Pat. No. 6,339,139 provided a gene transfer system binding to a growth factor receptor, comprising a 4-element complex gene transfer system consisting of ligand oligopeptide/polycationic polypeptide/endosome release oligopeptide/exogenous DNA or 3-element complex consisting of ligand oligopeptide/polycationic polypeptide/exogenous DNA.
The delivery vector systems in the art were confronted with one or more obstacles as described below. First, the immune responses are elicited by viral vector and cationic liposome when they are injected into living animals. Second, the extra-cellular fluids of cells, like blood stream, may dilute or eventually digest the gene cargo, which results in the lost of gene to be delivered. Third, the phospholipid bi-layer of cell membrane forms a natural barrier against the entrance of the nucleotide molecules. Therefore, the large molecules like DNA gene cannot cross the cell membrane in a freely (active or passive) transportation way to deliver the foreign DNA gene cargo into the cells. Fourth, the gel-like cytoplasm is rich in proteases and/or nucleases milieus in which the DNA gene cargo was degraded by the mechanism of endosomal trapping. Fifth, the vector-DNA cargo in the cytoplasm of cell meets the second barrier—nuclear membrane. The DNA gene cargo should pass the nuclear membrane and release the DNA gene cargo in the nucleus where the gene can act. Sixth, even if the DNA gene cargo has ability to enter the cell nucleus, the delivery efficiency is low.
Based on the above obstacles, there is a need to develop a delivery system for effectively delivering a desired molecule into cells or nuclei.
SUMMARY OF THE INVENTION
The invention relates to a fusion protein for delivery of a desired molecule into cells or nuclei, comprising i) a cold shock domain and the homologue or the functional equivalent derivatives thereof and ii) a membrane translocation sequence or the functional equivalent peptides and/or derivatives thereof.


REFERENCES:
patent: 6090619 (2000-07-01), Weissig et al.
patent: 6339139 (2002-01-01), Gu et al.
patent: 2390104 (2001-05-01), None
Becker-Hapak et al., “TAT-Mediated Protein Transduction into Mammalian Cells,”Methods24:247-256 (2001).
Fischer et al., “Cellular Delivery of Impermeable Effector Molecules in the Form of Conjugates with peptides Capable of Mediating Membrane Translocation,”Bioconjugate Chemistry12:825-841 (2001).
Ho et al., “Synthetic Protein Transduction Domains: Enhanced Transduction Potential in Vitro and in Vivo,”Cancer Research61:474-477 (2001).
Izumi et al., “Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'→5' exonuclease activity,”Nucleic Acids Research29:1200-1207 (2001).
Wang et al., “Acquisition of double-stranded DNA-binding ability in a hybrid protein betweenEscherichia coliCspA and the cold shock domain of humain YB-1,”Molecular Microbiology38:526-534 (2000).
Graumann, et al. “A superfamily of proteins that contain the cold-shock domain”, Tibs Trend in Biochemical Science, vol. 23, pp. 286-290 (1998).

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