Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-11-29
2002-12-17
Yucel, Remy (Department: 1636)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S009100
Reexamination Certificate
active
06495518
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention relates to biologically active molecules and to methods for delivery of biologically active molecules into the interior of cells by administering to the cells a complex comprising the molecule linked to a signal peptide. The present invention also relates to the development of cell-permeable peptide analogs and to methods for the targeted delivery of these peptide analogs to control systemic inflammatory response syndromes such as endotoxic shock.
2. Background Art
Peptides have been developed for many therapeutic uses. For example, diseases currently targeted by new peptide drugs include heart conditions, cancers, endocrine disorders, neurological defects, respiratory conditions, allergies and autoimmune diseases. Although the manufacture of known therapeutic peptides can be achieved by known methods, i.e., classic synthetic techniques or recombinant genetic engineering, delivery of the peptides into a cell has remained problematic, since they cannot readily cross biological membranes to enter cells. Thus, current methods include permeabilization of the cell membrane, or microinjection into the cell. Both of these methods have serious drawbacks. Permeabilization of cells, e.g., by saponin, bacterial toxins, calcium phosphate, electroporation, etc., can only be practically useful for ex vivo methods, and these methods cause damage to the cells. Microinjection requires highly skilled technicians (thus limiting its use to a laboratory setting), it physically damages the cells, and it has only limited applications as it cannot be used to treat for example, a mass of cells or an entire tissue, because one cannot feasibly inject large numbers of cells.
Similarly, delivery of nucleic acids has been problematic. Methods currently employed include the permeabilization described above, with the above-described drawbacks, as well as vector-based delivery, such as with viral vectors, and liposome-mediated delivery. However, viral vectors can present additional risks to a patient, and liposome techniques have not achieved satisfactorily high levels of delivery into cells.
Signal peptide sequences,
1
which share the common motif of hydrophobicity, mediate translocation of most intracellular secretory proteins across mammalian endoplasmic reticulum (ER) and prokaryotic plasma membranes through the putative protein-conducting channels.
2-11
Alternative models for secretory protein transport also support a role for the signal sequence in targeting proteins to membranes.
12-15
Several types of signal sequence-mediated inside-out membrane translocation pathways have been proposed. The major model implies that the proteins are transported across membranes through a hydrophilic protein conducting channel formed by a number of membrane proteins.
2-11
In eukaryotes, newly synthesized proteins in the cytoplasm are targeted to the ER membrane by signal sequences that are recognized generally by the signal recognition particle (SRP) and its ER membrane receptors. This targeting step is followed by the actual transfer of protein across the ER membrane and out of the cell through the putative protein-conducting channel (for recent reviews, see references 2-5). In bacteria, the transport of most proteins across the cytoplasmic membrane also requires a similar protein-conducting channel.
7-11
On the other hand, signal peptides can interact strongly with lipids, supporting the proposal that the transport of some secretory proteins across cellular membranes may occur directly through the lipid bilayer in the absence of any proteinaceous channels.
14-15
Thus, though many attempts have been made to develop effective methods for importing biologically active molecules into cells, both in vivo and in vitro, none has proved to be entirely satisfactory.
SUMMARY OF THE INVENTION
The present invention provides a method for treating or preventing sepsis in a human subject, comprising delivering to the subject a compound comprising a nuclear localization sequence of NF-&kgr;B such that nuclear importation of NF-&kgr;B is inhibited.
The present invention further provides a method of importing a biologically active molecule into a cell in a subject comprising administering to the subject a complex comprising the molecule linked to an importation competent signal peptide, thereby importing the molecule into the cell of the subject.
Additionally, the instant invention provides a method of importing a biologically active molecule into the nucleus of a cell in a subject comprising administering to the subject a complex comprising the molecule linked to an importation competent signal peptide and a nuclear localization peptide, thereby importing the molecule into the nucleus of the cell of the subject.
The present invention also provides a complex comprising an importation competent signal peptide linked to a biologically active molecule selected from the group consisting of a nucleic acid, a carbohydrate, a lipid, a glycolipid and a therapeutic agent.
REFERENCES:
patent: 5811512 (1998-09-01), Hirschmann et al.
patent: 5877282 (1999-03-01), Nadler et al.
patent: 5916872 (1999-06-01), Chang et al.
patent: 5962415 (1999-10-01), Nadler
patent: 6043339 (2000-03-01), Lin et al.
patent: WO98/11907 (1998-03-01), None
patent: wo-98/11907 (1998-03-01), None
Verma et al (1997) Nature 389:239-242.*
Palù et al. (1999) J. Biotechnol. 68:1-13.*
Luo et al (2000) Nature Biotechnology 18:33-37.*
Fox, ASM News, Feb. 2000, 66 (2): 1-3.*
Donnelly et al. “DNA Vaccines”, Annu. Rev. Immunol. 1997 15:617-48.*
Branch. “A good Antisense molecule is hard to find”, TIBS Feb. 23, 1998 pp. 45-50.*
Anderson. “Human gene therapy”, Nature vol. 392 1998 pp. 25-30.*
Qin et al. “Nuclear Factor kB contributes to excitotoxin-induced apoptosis in rat striatum”Mol. Pharmacology53:33-42 (1998).
Friedler et al. “Backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in nondividing cells”Biochemistry37:5616-5622 (1998).
Liu et al. “Identification of a functionally important sequence in the cytoplasmic tail of integrin Beta3 by using cell-permeable peptide analogs”Proc. Natl. Acad. Sci. USA93:11819-11824 (1996).
Zhang et al “Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules”Proc. Natl. Acad. Sci. USA95:9184-9189 (Aug. 1998).
Delli-Bovi et al. “An Oncogene Isolated by Transfection of Kaposi's Sarcoma DNA Encodes a Growth Factor that is a Member of the FGF Family” Cell, 50:729-737, Aug., 1987.
Delli-Bovi et al. “Processing, Secretion, and Biological Properties of a Novel Growth Factor of the Fibroblast Growth Factor Family with Oncogenic Potential” Mol. Cell. Biol. 8(7):2933-2941, Jul., 1988.
Wolff et al. “Nuclear Protein Import: Specificity for Transport across the Nuclear Pore” Exp. Cell Res. 178:318-334, 1988.
Dang and Lee “Nuclear and Nucleolar Targeting Sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat Proteins” J. Biol. Chem. 264(30):18019-18023, Oct. 25, 1989.
Landford et al. “Comparison of Diverse Transport Signals in Synthetic Peptide-Induced Nuclear Transport” Exp. Cell Res. 186:32-38, 1990.
Kieran et al. “The DNA Binding Subunit of NF-kB is Identical to Factor KBF1 and Homologous to the rel Oncogene Product” Cell 62:1007-1018, Sep. 1990.
Nolan et al. “DNA Binding and IkB Inhibition of the Cloned p65 subunit of NF-kB, a rel-Related Polypeptide” Cell 64:961-969, Mar. 8,1991.
Stochaj and Silver “A Conserved Phosphoprotein that Specifically Binds Nuclear Localization Sequences is Involved in Nuclear Import” J. Cell Biol. 117(3):473-482, May, 1992.
Lentz et al. “The E1 Replication Protein of Bovine Papillomavirus Type 1 Contains an Extended Nuclear Localization Signal That Includes a p34cdc2 Phosphorylation Site” J. Virol. 67(3):1414-14231, Mar., 1993.
Wessendorf et al. “Identification of a Nuclear Localization Sequence within the Structure of the Human Interleukin-1&agr;Precursor” J. Biol. Chem.
Collins Robert D.
Hawiger Jack J.
Liu Danya
Liu Xue Yan
Robinson Daniel
Loeb Bronwen M.
Needle & Rosenberg P.C.
Vanderbilt University
Yucel Remy
LandOfFree
Method for importing biologically active molecules into cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for importing biologically active molecules into cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for importing biologically active molecules into cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2919640