Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2002-01-07
2004-08-10
Falk, Anne-Marie (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S455000, C435S471000, C435S468000, C530S300000, C530S350000
Reexamination Certificate
active
06773920
ABSTRACT:
FIELD OF INVENTION
The present invention relates to methods for translocating polynucleotides and polypeptides between cells. More particularly, the present invention relates to use of translocating proteins to deliver a cell process-modifying molecule into the cell where the cell process-modifying molecule interacts specifically with a responsive target site.
BACKGROUND OF THE INVENTION
Translocating proteins are defined by their ability to cross biological membranes, such as cell membranes. A number of translocating proteins, have been described, including VP22 from Herpes Simplex Virus type 1 (G. Elliot and P. O'Hare,
Cell
88, 223-233 (1997)), a fragment of the Antennapedia protein from Drosophila (Antp) (D. Derossi et al.,
Journal of Biological Chemistry
269, 10444-10450 (1994)), and Protein H from
Streptococcus pyogenes
(Axcrona et al., Manuscript in preparation (1999)).
Antennapedia is a homeoprotein with a DNA binding domain composed of three alpha helices with a beta-turn separating helix 2 and 3. Experiments have demonstrated that a 16 amino acid peptide corresponding to the third helix, named Antp, can translocate across membranes and accumulate in the cytoplasm and nucleus (Derossi et al, supra). This peptide is internalized at a temperature as low as 4° C., suggesting that endocytosis is not responsible for the internalization of the peptide. In addition, since translocation does not require classical endocytosis, Antp does not travel through the endosomal and lysosomal compartments. Therefore, Antp is resistant to proteolysis and has enhanced activity in most cellular compartments (D. Derossi et al.,
J Biol Chem
271:18188-18193, 1996).
Recent experiments showing that a reverse helix (i.e. the reverse primary sequence) and a helix composed of D-enantiomers can transverse plasma membranes at 4° C. suggest that internalization of Antp involves the formation of inverted micelles in the phospholipid bilayer, making entry into cells receptor-independent and energy free (H. Hall et al.,
Current Biol
6:580-587, 1996).
The usefulness of Antp as a vector peptide has been proven successful by genetically fusing Antp to various peptides of interest (F. Perez et al.,
J Cell Sci
102:717-722, 1992; F. Perez et al.,
Mol Endocrinol
8:1278-87, 1994; and A. Prochiantz,
Curr Opinion Neurob
6:629-634, 1996) or by covalent linkage via cysteine residues (D. Derossi et al., supra). Internalization of peptides as large as 41 amino acids and of charged phosphopeptides (B. Allinquant et al.,
J Cell Biol
128:919-927, 1995) has been demonstrated in neuronal cells. In each case, the sequences fused to Antp retained their expected biological functions. Furthermore, Antp is the only translocating peptide that has been used to deliver oligonucleotides (up to 45 nucleotides in length) to cells in culture (C. M. Troy et al., J Neuroscience 16 253-61, 1996; G. Elliot et al., J Virol 72:6448-6455, 1998).
Protein H is a surface antigen of the human pathogen
Streptococcus pyrogenes.
Protein H is taken up by B- and T-lymphocytes and translocated to the nucleus. In contrast to other translocating proteins, which appear to have no effect on cellular function, protein H has a cytostatic effect thought to be the result of its association with the nuclear proteins SET and hnRNP A2/B1 (D. Derossi et al., supra). To date, the translocation of Protein H coupled to another molecule has not been demonstrated.
The best studied of the translocating proteins is the Herpes Simplex Virus protein VP22, which has the unique ability to translocate between cultured mammalian cells. When cells are transfected with a plasmid encoding the VP22 protein, the expressed protein accumulates in the cytoplasm of transfected cells and, by translocating across cell membranes, spreads to the surrounding non-transfected cells where it accumulates in the nuclei. This process can occur at 4° C. and also appears to be energy-free and independent of endocytosis. When protein trafficking though the cell is blocked using Brefeldin A, export of VP22 can still occur. Studies of cytoskeletal elements during VP22 trafficking suggest that the actin cytoskeleton may be involved in export or import of VP22 (Elliot and O'Hare, supra).
Delivery of several functional VP22 fusion proteins has been described, including VP22-p53 (A. Phelan et al.,
Nature Biotechnology
16:440-443, 1998)) and VP22-thymidine kinase (M. S. Dilber et al.,
Gene Therapy
6:12-21, 1999). At least twenty different mammalian cell types can take up a functional VP22-GFP fusion protein (Elliot and O'Hare, supra; Aints A., et al.,
J. Gene Med.
1:275-9, 1999; and Wybranietz W. A. et al.,
J. Gene Med.
1:265-274, 1999), including mouse skeletal myoblasts that are refractory to conventional transfection techniques (Derer W. et al.,
J. Mol. Med.
77: 609-6138, 1999).
Transfection of cells with plasmid DNA has been an invaluable tool for the study of biological systems. A variety of transfection methods (e.g. lipids, calcium phosphate) exist in the marketplace; however, these methods rarely result in more than 50% of cells expressing a gene carried on a plasmid with which the cells are transfected. Since most cells do take up exogenous DNA, inefficient transfections do not appear to be due to inability of the DNA complex to enter the cell. The majority of DNA is internalized by endocytosis with very little of the internalized DNA ever reaching the cytoplasm or nucleus where expression takes place. Indeed, observations of directly injected lipid-DNA complexes suggest that movement from the endosomes to the cytoplasm and nucleus is the most important limitation to successful transfections (J. H. Richardson et al.,
Proc. Natl. Acad. Sci.
92:3137-3141, 1995). Consistent with this observation, peptides with membrane fusion activity, like the fusogenic peptide of hemagglutinin (J. Zabner et al.,
Journal of Biological Chemistry
270:18997-9007, 1995), or a nuclear targeting sequence (M. Wilke et al.,
Gene Therapy
3, 1133-1142 (1996)) can increase transfection efficiencies in some cases.
Thus, there is a need in the art for new and better methods for modulating expression in cells of target genes and for transfection reagents and methods of their use to overcome the major blocks to expression of transfected genes, i.e., degradation in the endosomes and the inability of DNA to enter the cell nucleus.
BRIEF DESCRIPTION OF THE INVENTION
The present invention overcomes these problems in the art by providing method(s) for modulating a cellular process in a cell in culture by contacting such a cell with a cell process-modifying molecule attached to a translocating polypeptide under suitable conditions, whereby the cell process-modifying molecule is translocated into the cells in culture and interacts specifically therein with a target site responsive to the cell process-modifying molecule, thereby modulating a cellular process in the cell.
In another embodiment, the present invention provides method(s) for transfecting a cell in culture with a target gene by contacting the cell under suitable conditions with a polynucleotide comprising the target gene attached to a translocating polypeptide, whereby the cell is transfected with the target gene.
In still another embodiment, the present invention provides method(s) for modulating expression of a target gene product in a cell in culture that is transfected with the target gene under control of one or more regulatory elements by contacting the cell under suitable conditions with one or more regulatory agents attached to a translocating polypeptide, whereby the one or more regulatory agents are translocated into the cell and interact therein with the one or more regulatory elements, thereby modulating expression of the target gene product by the cell.
In yet another embodiment, the present invention provides vector(s) comprising a polynucleotide encoding a cell process-modifying molecule attached to a translocating polypeptide.
REFERENCES:
patent: 4959217 (1990-09-01), Sanders et al.
patent: 5328984 (1994-07-
Bennett Robert P.
Dalby Brian
Falk Anne-Marie
Greenlee Winner and Sullivan P.C.
Invitrogen Corporation
Sullivan Daniel M.
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