Stock material or miscellaneous articles – Coated or structually defined flake – particle – cell – strand,... – Particulate matter
Reexamination Certificate
1998-07-06
2001-06-12
Brusca, John S. (Department: 1631)
Stock material or miscellaneous articles
Coated or structually defined flake, particle, cell, strand,...
Particulate matter
C435S458000, C530S363000, C530S350000, C536S023100, C424S009321
Reexamination Certificate
active
06245427
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to intracellular substance delivery the intracellular delivery of biologic molecules. More specifically, it relates to the use of liposomal complexes for intracellular delivery of biologically active agents.
BACKGROUND OF THE INVENTION
One of the major problems of gene therapy is the effective delivery of the therapeutic agent into target cells in vitro or in vivo. Although viral vectors have certain advantages, including high levels of transfection, or efficient and stable integration of foreign DNA into a wide range of host genomes, they suffer from several problems including immunogenicity, toxicity, difficulty of large-scale production, size limit of the exogenous DNA, random integration into the host genome, and the risks of inducing tumorigenic mutations and/or generating active viral particles through recombination (Singhal, A. and Huang, L., (1994) In: Wolf, J. A.(ed),
Gene Therapeutics: Methods and Applications of Direct Gene Transfer
. Birkhauser: Boston, pp118-142: Lee, R. J., and L. Huang, (1996)
J. Biol. Chem
. 271:8481-8487). These limitations of viral vectors have prompted investigators to try to improve methods of non-viral gene delivery. (Treco, D. A. and R. F. Selden, (1995)
Mol. Med. Today
1:314-321).
Cationic liposomes have been used extensively for in vitro and in vivo gene delivery, and constitute a viable alternative to viral gene delivery vehicles. (Singhal A., and L. Huang, supra; Hug P and R. G. Sleight, (1991)
Biochim Biophys Acta
1097: 1-17: Lasic D. D. and N. S. Templeton, (1996)
Adv. Drug Deliv. Rev
. 20: 221-266). Using this delivery system, relatively stable expression has been achieved in a number of tissues. (Liu, Y., et al., (1995)
J Biol Chem
270: 24864-24870: Thierry, A. R., et al., (1995)
Proc Natl Acad Sci USA
92: 9742-9746: Takehara, T., et al., (1995)
Hepatology
21:46-751.)
The efficiency of cationic liposome transfection was improved through the use of two different approaches. The first approach was based on the promotion of cellular internalization of the cationic liposome-DNA complexes through receptor-mediated endocytosis. For this purpose iron-saturated human transferrin, and other receptor-mediated ligands, were associated with the lipid-DNA complexes, referred to herein as “lipoplexes” (Felgner,et al., (1997)
Hum. Gene Ther
. 8: 511-512) at different (+/−) charge ratios. Transferrin is a useful ligand that binds to a cell-surface receptor expressed by most proliferating cells, with particularly high expression on erythroblasts and tumor cells (Wagner et al., (1994)
Adv. Drug. Del. Rev
. 14:113-136). Another report indicated that associating transferrin with cationic liposomes also enhanced transfection of HeLa cells (Cheng, P. W. (1996)
Hum. Gene Ther
. 7:275-282). The use of transferrin as a receptor-ligand in lipoplex-mediated transfections was also disclosed by Cheng, P. W. in PCT publication WO 97/28817, incorporated herein by reference is referred to hereinafter as “Cheng (1997)”.
The second approach was based on the association of endosome disrupting agents to the lipoplexes with the purpose of facilitating the cytoplasmic release of DNA from endosomes, thus preventing its lysosomal degradation and therefore enhancing transfection. Two different synthetic fusogenic peptides, “GALA” and the influenza virus hemagglutinin HA2 N-terminal peptide (hereinafter, “HA-2”), both low pH-activated rrtmbrane-active peptides, were used for that purpose (Simoes, S. et al.(1998)
Gene Ther
., in press).
The use of receptor-specific ligands as a targeting protein in liposomal delivery vehicles in vitro and in vivo presents several problems. Receptor-specific ligands are relatively rare molecules and incur considerable expense in isolating and collecting an adequate supply. Receptor-ligands are invariably potent effectors of biological response. Use of such molecules in lipoplexes increase their concentration in the cellular milieu and therefore pose a potentially serious threat of adverse or unwanted side-effects. Finally, studies have shown that high concentrations of blood serum can inhibit the efficiency of ligand-mediated lipoplexes, thereby raising serious questions as to their utility in an in vivo environment. (Cheng (1997).
SUMMARY OF THE INVENTION
This invention describes unique liposomal complexes for the intracellular delivery of particular agents or molecules such as DNA, RNA or proteins. This invention also describes methods for the use of these complexes for intracellular delivery. A unique feature of this invention is that it utilizes polypeptides that lack cell-receptor specificity (non-ligands, also referred to as non-receptor-binding polypeptides) combined with cationic liposomes as a delivery vehicle. Molecules desirable for intracellular delivery include, but are not limited to DNA, polydeoxyribonucleotides, RNA, polyribonucleotides, proteins, peptides, polypeptides and the like.
It is an object of this invention to provide carrier compositions for intracellular delivery of a biologic molecule or agent.
It is an object of this invention to provide carriers that do not include viral components.
It is an object of this invention to provide carriers that do not utilize receptor-ligands or receptor-mediated transport molecules.
It is an object of this invention to provide carriers that comprise liposomes and serum proteins that bind to cells non-specifically.
It is an object of this invention to utilize vehicle components that are abundant, inexpensive and easy to isolate in large quantity.
It is an object of this invention to provide a vehicle whose components are not likely to cause an undesirable biological reaction.
It is an object of this invention to provide a vehicle that is effective in the presence of high concentrations of blood serum.
It is an object of this invention to provide methods of using carrier cdmpositions to effect intracellular delivery of particular molecules.
In accordance with the above objects and other described herein, the carrier composition for intracellular delivery of a biologically active molecule of the present invention comprises a mixture of a cationic lipid and a non-receptor-binding protein.
In another embodiment of the invention the cationic lipid is combined with a neutral lipid forming a cationic liposome thereby.
In another embodiment, the carrier composition comprises a cationic lipid and serum albumin of human or non-human origin.
In another embodiment, the carrier composition comprises a cationic lipid, a protein that lacks receptor specificity (a non-receptor-binding protein) and a biologically active molecule.
In further accordance with the above objects and others described herein, the method for intracellular delivery of a biologically active agent to a target cell comprises:
a) combining a non-receptor-binding polypeptide and a cationic lipid to form a first mixture such that said polypeptide and cationic lipid become associated;
b) adding to said first mixture said biologically active agent to form a second mixture such that said agent becomes associated with said cationic lipid; and,
c) introducing said second mixture to said cell.
In another embodiment of the method, the non-receptor-binding polypeptide is a protein. In a preferred embodiment, the protein is a serum albumin, most preferably, the serum albumin of the animal source of the target cell to be inoculated.
In another embodiment of the method, the cationic lipid is combined with a neutral lipid forming a cationic liposome thereby.
REFERENCES:
patent: 5521291 (1996-05-01), Curiel et al.
patent: 5547932 (1996-08-01), Curiel et al.
patent: WO 97/28817 (1997-08-01), None
Simoes et al. Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum. Biochimica Biophysica Acta vol. 1463 pp. 459-469, 2000.*
Singhal A, Huang L, Gene transfer in mammalian cells using liposomes as carriers, Gene Therapeutics: Methods and Applications of Direct Gene Transfer, In: Wolf JA (ed) Birkhäuser: Boston, p
Düzgünes Nejat
Pedras de Lima Maria C.
Simões Sérgio
Slepushkin Vladimir
Brusca John S.
Dolberg, Esq. David
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