Lipidic microparticles linked to multiple proteins

Details Lipidic microparticles linked to multiple proteins Lipidic microparticles linked to multiple proteins

2002-06-21

2004-10-12

Ketter, James (Department: 1636)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C530S411000, C530S350000

Reexamination Certificate

active

06803053

ABSTRACT:

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
FIELD OF THE INVENTION
The present invention relates to the field of cationic lipid:DNA complexes (“CLDC”). In particular, the present invention relates to lipid:nucleic acid complexes that contain (1) hydrophilic polymer; (2) nucleic acid that has been condensed with organic polycations; and (3) hydrophilic polymer and nucleic acid that has been condensed with organic polycations. The lipid:nucleic acid complexes of this invention show high transfection activity in vivo following intravenous injection and an unexpected increase in shelf life, as determined by in vivo transfection activity.
The present invention further relates to the field of lipidic microparticles, such as liposomes, lipid:DNA complexes, lipid:drug complexes, and microemulsion droplets, attached to proteins. In particular, the invention relates to lipidic microparticles with attached proteins which have been first conjugated to linker molecules having a hydrophilic polymer domain and a hydrophobic domain capable of stable association with the microparticle, or proteins which have been engineered to contain a hydrophilic domain and a lipid moiety permitting stable association with a lipidic microparticle.
BACKGROUND OF THE INVENTION
Liposomes that consist of amphiphilic cationic molecules are useful non-viral vectors for gene delivery in vitro and in vivo (reviewed in Crystal,
Science
270: 404-410 (1995); Blaese et al.,
Cancer Gene Ther.
2: 291-297 (1995); Behr et al.,
Bioconjugate Chem.
5: 382-389 (1994); Remy et al.,
Bioconjugate Chem.
5: 647-654 (1994); and Gao et al.,
Gene Therapy
2: 710-722 (1995)). In theory, the positively charged liposomes complex to negatively charged nucleic acids via electrostatic interactions to form lipid:nucleic acid complexes. The lipid:nucleic acid complexes have several advantages as gene transfer vectors. Unlike viral vectors, the lipid:nucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size. Since the complexes lack proteins, they may evoke fewer immunogenic and inflammatory responses. Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency.
There are a number of publications that demonstrate convincingly that amphiphilic cationic lipids can mediate gene delivery in vivo and in vitro, by showing detectable expression of a reporter gene in culture cells in vitro (Felgner et al.,
Proc. Natl. Acad. Sci. USA
84: 7413-17 (1987); Loeffler et al.,
Methods in Enzymology
217: 599-618 (1993); Felgner et al.,
J. Biol. Chem.
269: 2550-2561 (1994)). Because lipid:nucleic acid complexes are on occasion not as efficient as viral vectors for achieving successful gene transfer, much effort has been devoted in finding cationic lipids with increased transfection efficiency (Behr,
Bioconjugate Chem.
5: 382-389 (1994); Remy et al.,
Bioconjugate Chem.
5: 647-654 (1994); Gao et al.,
Gene Therapy
2: 710-722 (1995)). Lipid:nucleic acid complexes are regarded with enthusiasm as a potentially useful tool for gene therapy.
Several groups have reported the use of amphiphilic cationic lipid:nucleic acid complexes for in vivo transfection both in animals, and in humans (reviewed in Gao et al.,
Gene Therapy
2: 710-722 (1995); Zhu et al.,
Science
261: 209-211 (1993); and Thierry et al.,
Proc. Natl. Acad. Sci. USA
92: 9742-9746 (1995)). However, the technical problems for preparation of complexes that have stable shelf-lives have not been addressed. For example, unlike viral vector preparations, lipid:nucleic acid complexes are unstable in terms of particle size (Behr,
Bioconjugate Chem.
5: 382-389 (1994); Remy et al.,
Bioconjugate Chem.
5: 647-654 (1994); Gao et al.,
Gene Therapy
2: 710-722 (1995)). It is therefore difficult to obtain homogeneous lipid:nucleic acid complexes with a size distribution suitable for systemic injection. Most preparations of lipid:nucleic acid complexes are metastable. Consequently, these complexes typically must be used within a short period of time ranging from 30 minutes to a few hours. In recent clinical trials using cationic lipids as a carrier for DNA delivery, the two components were mixed at the bed-side and used immediately (Gao et al.,
Gene Therapy
2: 710-722 (1995)). The structural instability along with the loss of transfection activity of lipid:nucleic acid complex with time have been challenges for the future development of lipid-mediated gene therapy.
Liposomes consisting of amphiphilic cationic molecules are not, of course, the only form of lipidic microparticles and gene therapy is not the only utility for such particles. Lipidic microparticles have also been used for delivery of drugs and other agents to target sites. Targeting of the microparticles is typically achieved through use of a protein attached to the surface of the microparticle, which may, for example be a ligand for cell surface receptor on a cell type of interest. Conversely, the protein may be an antibody which specifically recognizes an antigen on a cell type of interest, such as diseased cells carrying specific markers. Additionally, proteins can be attached for purposes other than targeting. For example, liposomes can contain prodrugs which slowly seep from the liposome into the circulation. An enzyme attached to the liposome can then convert the prodrug into its active form.
Current methods for effecting the attachment of proteins to lipidic microparticles have been of two types. The first type requires introducing a linker molecule bearing an “active” group (one which reacts with a functional group of the protein) into the microparticle composition prior to conjugation of the “activated” particle with the protein of interest. The disadvantages of methods of this type are: often uncontrollable, incomplete reaction of the protein with the linker; the presence of excess linker on the resulting conjugate, potentially adverse effect of the linker on the ability of the particle, and the inability to incorporate components reactive with the links into the composition of the particle.
The second group of methods employs the steps of (a) attachment of a hydrophobic moiety, such as a hydrocarbon chain, to the protein molecule, (b) dissolving the components of the lipidic microparticle, along with the conjugate of step (a) in the presence of a detergent, and (c) removing the said detergent, effecting the formation of the lipidic particle incorporating the protein conjugate (Torchilin,
Immunomethods
4-244-258 (1994); Laukkanen et al.,
Biochemistry
33:11664-11670 (1994)). These methods have a number of disadvantages, including the imposition of severe limitations on the range of methods by which the particle can be formed, (e.g. the detergent removal technique is required) and by which the drug or other agent can be loaded into the microparticle. Moreover, step (b) requires the dissolution of the microparticle. These methods are therefore unable to attach a protein to a premade particle without first destroying it. The presence of detergent in these methods is unavoidable because without a detergent the hydrophobically modified protein is insoluble in aqueous medium
The “insertion” into liposomes of hydrophilic polymer-lipid linked to a small (5 amino acid) oligopeptide or small oligosaccharide has been reported. (Zalipsky et al.,
Bioconjugate Chem.
8:111-118 (1997). The peptide and oligosaccharide employed were, however, of a size (molecular weight, 500-3,000 Da) smaller than, or comparable to, the linker itself (molecular weight 2,750 Da). This study therefore provides no guidance for inserting into liposomes or other lipidic microparticles proteins, such as antibodies, or fragments thereof, conjugated to linkers significantly smaller than the protein. In view of the hydrophilic nature of antibodies and other proteins, the art has taught that the larger, protein portion of such a conjugate prevents the hydrophobic linking moiety from stable association with a lipidic microparticle.
SUMMARY OF THE INVENTION
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