Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1996-01-08
2002-02-05
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S345000, C530S359000, C530S402000
Reexamination Certificate
active
06344436
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of delivering agents, including genes and other biological macromolecules, to cells.
BACKGROUND OF THE INVENTION
The following description of the background of the invention is provided to aid in understanding the claimed invention, but it is not admitted to constitute or describe prior art to the claimed invention and should in no way be construed as limiting the claimed invention.
Several techniques currently exist for delivering genes to cells and many clinical trials are currently ongoing in order to evaluate the degree of therapeutic efficacy obtained using such methods. One method of gene delivery involves the use of recombinant retroviral vectors for delivery of genes to cells of living animals. Morgan et al.,
Annu. Rev. Biochem.,
62:191-217 (1993). Retroviral vectors permanently integrate the transferred gene into the host chromosomal DNA. In addition to retroviruses, other virus have been used for gene delivery. Adenoviruses have been developed as a means for gene transfer into epithelial derived tissues. Stratford-Perricaudet et al.,
Hum. Gene. Ther.,
1:241-256 (1990); Gilardi et al.,
FEBS,
267:60-62 (1990); Rosenfeld et al.,
Science,
252:4341-4346 (1991); Morgan et al.,
Annu. Rev. Biochem.,
62:191-217 (1993). Recombinant adenoviral vectors have the advantage over retroviruses of being able to transduce nonproliferating cells, as well as an ability to produce purified high titer virus.
In addition to viral-mediated gene delivery, a more recent means for DNA delivery has been receptor-mediated endocytosis. Endocytosis is the process by which eucaryotic cells continually ingest segments of the plasma membrane in the form of small endocytotic vesicles. Alberts et al.,
Mol. Biol. Cell,
Garland Publishing Co., New York, 1983. Extracellular fluid and material dissolved in it becomes trapped in the vesicle and is ingested into the cell. Id. This process of bulk fluid-phase endocytosis can be visualized and quantified using a tracer such as enzyme peroxidase introduced into the extracellular fluid. Id.
Taking advantage of receptor-mediated endocytosis, the asialoglycoprotein receptor has been used in targeting DNA to HepG2 cells in vitro and liver cells in vivo. Wu et al.,
J. Biol. Chem.,
262:4429-4432 (1987); Wu et al.,
Bio.,
27:887-892 (1988); Wu et al.,
J. Biol. Chem.,
263:14620-14624 (1988); Wu et al.,
J. Biol. Chem.,
264:16985-16987 (1989); Wu et al.,
J. Biol. Chem.,
266:14338-14342 (1991). These studies used asialoorosomucoid covalently linked to polylysine with water soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or with 3′(2′pyridyl-dithio)propionic acid n-hydroxysuccinimide ester. Polylysine in the studies above bound DNA through ionic interaction. The DNA was ingested by endocytosis.
Other studies have utilized transferrin and the transferrin receptor for delivery of DNA to cells in vitro. Wagner et al.,
P.N.A.S.,
87:3410-3414 (1990). These studies modified transferrin by covalently coupling transferrin to polylysine. Id. The polylysine interacted ionically with DNA. Delivery of DNA occurred to cells through the transferrin receptor. Such analyses were performed in vitro. Id. Cotten et al.,
P.N.A.S.,
87:4033-4037 (1990); Zenk et al.,
P.N.A.S.,
87:3655-3659 (1990).
In addition to delivery DNA (Gottschalk et al.,Gene Therapy 1:185-91 (1993)), other macromolecules can also be delivered by receptor-ligand systems. Leamon et al.,
P.N.A.S.,
88:5572-5576 (1991); Leamon et al.,
J. Biol. Chem.,
267
:
24966
-
24971
(1992). In particular these studies have involved the folate receptor, an anchored glycosyl-phosphatidyl protein, which is excluded from coated pits and cycles in and out of the cells by caveolae. Anderson et al.,
Science,
252:410-411 (1992). This uptake mechanism has been called potocytosis. Id. Folate conjugated enzymes have been delivered into cells through this receptor system and retained activity for at least six hours. Leamon et al.,
P.N.A.S.,
88:5572-5576 (1991). Folate receptors have limited tissue distribution and are overexpressed in several malignant cell lines derived from many tissues. Weitman et al.,
Cancer Res.,
52:3396-3401 (1992); Weitman et al.,
Cancer Res.,
52:6708-6711 (1992); Campbell,
Cancer Res.,
51:5329-5338 (1991); Coney,
Cancer Res.,
51:6125-6123 (1991). Other studies have also used biotin or folate conjugated to proteins by biotinylation for protein delivery to the cell. Low et al., U.S. Pat. No. 5,108,921.
Nucleic Acid Transporters for Delivery of Nucleic Acids into a Cell; Smith et al., U.S. patent application Ser. No. 08/484,777, filed Dec. 18, 1995, incorporated herein by reference in its entirety including any drawings.
A non-exchangeable apolipoprotein E peptide that mediates binding to the low density lipoprotein receptor is described in Mims et al., Journal of Biological Chemistry, 269 (32) 20539-20547, 1994, incorporated herein by reference in its entirety, including any drawings.
SUMMARY OF THE INVENTION
The present invention provides novel uses of lipophilic peptides for delivering macromolecules (e.g. nucleic acids) into a cell, complexes formed between the macromolecules to be delivered and the lipophilic peptide, and cells transformed by such complexes. Thus, the present invention allows for enhanced delivery of macromolecules (including nucleic acids) into cells.
The lipophilic peptide has a delivery peptide portion and a lipid moiety portion. The amino acid sequences of several suitable delivery peptides are set forth herein and those skilled in the art would be able to make and use many others given the methods described herein. The lipid moiety makes the delivery peptide lipophilic and examples of suitable modifications are provided herein. Again, however, those skilled in the art would be able to make and use lipophilic peptides having different lipid moieties.
Thus, in a first aspect, the present invention features a peptide-macromolecule complex for delivering a macromolecule into a cell. The complex includes a lipophilic peptide having a delivery peptide associated with a lipid moiety. The delivery peptide portion of the lipophilic peptide is complexed to the macromolecule.
The term “peptide-macromolecule complex” as used herein refers to a molecular complex which is capable of transporting a macromolecule through the cell membrane. This molecular complex is preferably bound to a macromolecule noncovalently. The peptide-macromolecule complex should be capable of transporting nucleic acid in a stable and condensed state and of releasing the noncovalently bound nucleic acid into the cellular interior. Furthermore, the nucleic acid carrier may prevent lysosomal degradation of the nucleic acid by endosomal lysis. In addition, although not necessary, the peptide-macromolecule complex can also efficiently transport the nucleic acid through the nuclear membrane, as discussed below.
The peptide-macromolecule complex as described herein can contain, but is not limited to, seven components. It comprises, consists or consists essentially of: (1) a nucleic acid or other macromolecule with a known primary sequence that contains the genetic information of interest or a known chemical composition; (2) a peptide agent capable of stabilizing and condensing the nucleic acid or macromolecule in (1) above; (3) an N termini acylation moiety to increase the lipophilicity of the peptide agent in (2) above (4) a lysis moiety that enables the transport of the entire complex from the cell surface directly into the cytoplasm of the cell; (5) a moiety that recognizes and binds to a cell surface receptor or antigen or is capable of entering a cell through cytosis; (6) a moiety that is capable of moving or initiating movement through a nuclear membrane; and/or (7) a nucleic acid or macromolecular molecule binding moiety capable of covalently binding the moieties of (2), (3), (4), (5), and (6) above.
The term “delivery” refers to transportation of a molecule to a desired cell or any cell. Deli
Hauer Jochen
Mims Martha P.
Smith Louis C.
Sparrow James T.
Baylor College of Medicine
Carlson Karen Cochrane
Lyon & Lyon LLP
Tu Stephen
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