Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2002-07-02
2004-11-23
Russel, Jeffrey Edwin (Department: 1654)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S013800, C514S016700, C514S04400A, C530S300000, C530S326000, C530S329000, C530S345000, C530S408000, C530S409000, C530S410000, C536S023100
Reexamination Certificate
active
06821948
ABSTRACT:
The present invention relates to conjugates for mediating cell-specific, compartment-specific or membrane-specific transport of active substances. The invention also relates to methods of producing said conjugates and their use.
As is known, cellular membrane systems are largely impermeable to many substances (e.g. nucleic acids, proteins, chemical substances) which shall be introduced into a cell from outside. For the introduction of nucleic acids it is possible to penetrate cell membranes by physical processes (transfection in the case of eukaryotes, transformation in the case of prokaryotes) and biological processes (infection). In the case of transformation, i.e. the direct take-up of the naked nucleic acid by the cell, the cells are treated beforehand. Various methods are available to produce these “competent cells”. Most methods are based on the observations made by Mandel and Higa (J. Mol. Biol. 53, pages 159-163 (1970)) who were the first to show that it is possible to substantially increase the yields occurring when lambda-DNA is taken up by bacteria in the presence of calcium chloride. This method was used successfully for the first time by Cohen et al. (Proc. Natl. Acad. Sci. U.S.A. 69, pages 2210-2114 (1972)) for plasmid DNA and has been improved by many modifications. Another transformation method is based on the observation that high-frequency alternating-current fields can break up cell membranes (electroporation). This technique can be utilized to insert naked DNA not only in prokaryotic cells but also in eukaryotic cell systems (Weaver et al., J. Cell Biochem. 51, pages 426-435 (1993)). Two very mild methods of introducing DNA into eukaryotic cells were developed by Sikes et al. (Hum. Gen. Therap. 5, pages 837-840 (1994)) and Yang et al. (Proc. Natl. Acad. Sci U.S.A. 87, pages 9568-9572 (1990). They are based on the direct injection of the DNA into single cells (microinjection) and on the bombardment of a cell population using microprojectiles of tungsten on the surface of which the corresponding nucleic acid was bound (gene gun), respectively. In a progress parallel to the physical transformation of cells, biological infection methods have proved their efficiency. They comprise in particular the viral introduction of nucleic acids into cells (Chatterjee et al., Science 258, pages 1485-1486 (1992); Cossett and Rusell, Gene Therapy 3, pages 946-956 (1996); Bilbao et al., FASEB J. 11, pages 624-634 (1997)) and the liposome-mediated lipofection (Bennett et al., J. Drug Targeting 5, pages 149-162 (1997)). Reference is also made to standard methods of the liposomal transport (Gao and Huang, Gene Therapy 2, pages 710-722 (1995); Akhtar et al., Nucl. Acid. Res. 19, pages 5551-5559 (1991)) and poly-L-lysine formation (Leonetti et al., Bioconj. Chem. 1(2), page 149 (1990) of active substances to be able to transport them into cells.
Despite the above-listed plurality of methods of passing through the cellular membrane systems, there is no universal method serving for introducing different active substances into cells. All of the above-mentioned physical and biochemical methods are artificial and non-physiological unless they make use of cell-immanent mechanisms. It is presently not yet certain that viruses used as transport vehicles are free of toxicity. They are often not effective and, in addition, they are detected by the immune system.
It was therefore the object of the present invention to provide a possibility of permitting the site-directed and specific introduction of active substances into cells and compartments. The following demands must be complied with in this connection:
universal applicability
cell-specific, compartment-specific and membrane-specific introduction behavior
high degree of effectiveness
low immunogenicity
minimization of the infection risk
sufficiently long residence time.
This object is achieved by the subject matters defined in the claims.
The inventors developed a conjugate comprising the following components:
a transport mediator for the cell membrane (“P”),
a cell-specific, compartment-specific or membrane-specific address protein or peptide (“AP”), and
an active substance to be transported (“W”).
The conjugate according to the invention is preferably composed as follows:
P-AP-W
More preferably it comprises a spacer (“SP”):
P-AP-SP-W
The transport mediator for the cell membrane (abbreviated as “P” above) is a peptide or protein which can penetrate the plasma membrane. The length of this peptide or protein is not subject to limitation as long as it has the above property. Examples of “P” are derived preferably from the penetratin family (Derossi et al., 1998
, Trends Cell Biol.
8, pages 84-87) or are transportan or parts thereof (Pooga et al., The Faseb Journal (1998), Vol. 12, page 68 et seq.), those of the penetratin family being preferred. An example of “P” is a penetratin having the following sequence:
NH
2
-RQI KIWFQNRRMKWKK-(SEQ ID NO.: 1)
(NH2-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys Trp-Lys-Lys)
Further examples of the transport protein “P” are as follows:
Viral transport protein
PTD protein transduction domain (TAT/HIV-1)
1—letter code H
2
N-YGRKKRRQRRR-COOH (SEQ ID NO: 12)
3-letter code H
2
N-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-
Bacterial transport molecule
TP protein transport domain TP(Eco)
1-letter code H
2
N-MTRQTFWHRIKH-CQOH (SEQ ID NO: 13)
3-letter code H2N-Met-Thr-Arg-Gln-Thr-Phe-Trp-His-Arg-Ie-Lys-His
The select “P” sequence is produced biologically (purification of natural transport mediator proteins or cloning and expression of the sequence in a eukaryotic or prokaryotic expression system), preferably synthetically, e.g. according to the established Merrifield method (Merrifield, J. Am. Chem. Soc. 85: 2149, 1963).
The selection of the address protein or peptide (abbreviated as “AP” above) depends on the membrane or membrane system which has to be penetrated and the target compartment of the cell (cytoplasm, nucleus, mitochondria, chloroplast, endoplasmic reticulum) or the cell organelle which shall be reached. The length of this address peptide or protein is not subject to limitation as long as it comprises the property of ensuring a cell-specific, compartment-specific or membrane-specific transport. For the introduction of active substances, in particular nucleic acids, “APs” are generally used which contain a cell-specific, compartment-specific or membrane-specific recognition signal, directing the attached active substance to its site of action. There are the “APs” to chose from which can transport active substances in the presence or absence of a membrane potential. The pure address sequence is usually sufficient for a transport into the cell compartment. However, it is also possible to chose “APs” which have a cell-specific or compartment-specific peptidase cleavage site. In the most favorable case, this cleavage site lies within the signal sequence but it can also be attached thereto by additional amino acids to ensure the cleavage of the address sequence after the target compartment is reached. The select “AP” sequence is produced biologically (purification of natural transport mediator proteins or cloning and expression of the sequence in a eukaryotic or prokaryotic expression system), preferably synthetically, e.g. according to the established Merrifield method (Merrifield, J. Am. Chem. Soc. 85: 2149, 1963). Examples of address proteins or peptides are as follows:
Import into the ER H
3
N+-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gin-(SEQ ID NO: 2);
Reimport into the ER H
2
N-Lys-Asp-Glu-Leu-COO
−
(SEQ ID NO: 3);
Import into the mitochondria H
3
N+-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu-(SEQ ID NO: 4);
Import into the nucleus—Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val (SEQ ID NO: 5);
H
3
N+-Pro-Lys-Lys-Lys-Arg-Lys-Val-(=nuclear localisation sequence from 5V40-T antigen) (SEQ ID NO: 6);
Import into peroxisomes H
2
N-Ser-Lys-Leu-COO
−
Braun Klaus
Debus Jürgen
Friedrich Eckart
Peschke Peter
Pipkorn Rüdiger
Deutsche Krebsforschungszentrum Stiftung des Offentlichen Rechts
Fuierer Marianne
Hultquist Steven J.
Russel Jeffrey Edwin
Yang Yongshi
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