Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-17
2004-02-17
Lacourciere, Karen (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091100, C435S440000, C435S455000, C435S456000, C435S458000, C435S325000, C424S450000
Reexamination Certificate
active
06692911
ABSTRACT:
BACKGROUND OF THE INVENTION
The recent revolutions in molecular and pharmaceutical biology and pharmaceutical chemistry have created a need for the development of effective mechanisms for delivering biological and other therapeutic agents into cells. Researchers have particularly struggled to develop an efficient means of introducing nucleic acids into cells, for example for gene therapy, antisense therapy, or research purposes (e.g., to study cell differentiation, growth and carcinogenic transformation or to create animal models for human disease; see, for example, Abdallah,
Biol. Cell,
85:1, 1995 and references therein).
Unfortunately, existing techniques for delivering nucleic acids to cells are limited by poor efficiency and/or high toxicity of the delivery reagents. A particular problem is encountered with techniques that rely on receptor-mediated endocytosis (see, e.g.,
FIG. 1
) because the nucleic acid to be delivered is often destroyed when exposed to the low pH and active degradatory machinery of the endosome/lysosome. Various reagents (e.g., chloroquine, polyethylenimine [PEI], certain highly charged cationic compounds, fusogenic peptides, and inactivated adenoviruses) have been developed that are intended to quickly disrupt the endosome in order to minimize the amount of time that a delivered nucleic acid spends in this hostile environment.
Certain of these known compounds (i.e., chloroquine, PEI), are thought to act as so-called “proton-sponges” because they contain a large number of proton-acceptor sites. It is thought that these compounds sop up protons in the endosome, thereby increasing the pH in the endosome (see, for example, Boussif et al.,
PNAS,
92:7297, 1995). This pH increase both inhibits the action of lysosomal nucleases with acid-optimal pH dependence and induces an ATPase proton pump in the endosomal membrane to furiously pump additional protons from the cytoplasm into the endosome in order to restore the proper endosomal pH. Because the ATPase pump carries one chloride ion into the endosome with every proton that it transfers from the cytoplasm, its excessive pumping creates an osmotic pressure imbalance that results in lysis of the endosome (see Behr, ILMAC, 1st Swiss Cost Chemistry Symposium, 1996; see also FIG.
2
).
The highly charged cationic compounds are thought to burst open the endosomal compartment by a different mechanism that involves fusing with and lysing open the bilayer membranes. The fusogenic peptides and inactivated viruses rely on viral lysis capabilities to burst the endosome compartment.
Although these known endosomolytic agents do appear to increase the efficiency of nucleic acid delivery, they have serious toxicity problems and other disadvantages. Some (e.g., chloroquine) are simply poisonous to cells. Others (e.g., viral compounds) can activate the immune system, thereby risking systemic difficulties and also creating the possibility that the host immune system will destroy the agent relied upon to effect cell delivery. There remains a need for the development of a biocompatible, preferably biodegradable, endosomolytic cell delivery agent. There is a particular need for an agent that can efficiently introduce nucleic acids into cells.
SUMMARY OF THE INVENTION
The present invention provides improved cell delivery compositions. In particular, the invention provides a biocompatible endosomolytic system. These inventive endosomolytic agents obviate the need for known agents (i.e., chloroquine, fusogenic peptides, inactivated adenoviruses, and polyethyleneimine) that can burst endosomes but have negative effects on cells. Preferred inventive endosomolytic agents are biodegradable in that they are broken down within cells into components that the cells can either reuse or dispose of. Particularly preferred inventive endosomolytic agents are cationic polymers comprised of biomolecules. Although the present invention is not limited by the mechanism of action of the endosomolytic agents, certain preferred agents have multiple proton acceptor sites and would be expected to act as “proton sponges”, disrupting the endosome by osmolytic action. Particularly preferred agents are polycationic under the conditions of the endosome (i.e., at pH 4). Exemplary endosomolytic agents include, but are not limited to, imidazole containing compounds such as histidine, histamine, vinylimidazole, polymers thereof, and any combinations thereof.
In one preferred embodiment of the invention, the endosomolytic agent comprises polyhistidine. Polyhistidine for use in accordance with the present invention may be provided as a linear or branched polyhistidine polymer. Moreover, as is discussed further below, the polyhistidine may be provided in combination with one or more additional agents. Where such other agents are other polymers, or functionalizable chemical compounds, they may be co-polymerized or functionalized with polyhistidine or histidine. Thus, a polyhistidine endosomolytic agent of the present invention need not comprise a polyhistidine polymer per se, so long as it has a sufficient number of histidine functional groups to preserve poyhistidine functionality as described herein. To give but one example, the inventive endosomolytic agent may comprise a single linear or branched copolymer synthesized from any appropriate combination of polyhistidine, polylysine, histidine, and/or lysine.
The endosomolytic agents of the present invention may be employed in any of a variety of delivery contexts. In some cases, the endosomolytic agent also acts as a delivery agent; in other cases, the endosomolytic agent is combined with a delivery agent that complexes the compound being delivered in a manner that allows that compound to be taken into an endosome and thereby introduced into a cell. Thus, the present invention also provides a cell delivery system comprising an endosomolytic agent, a delivery agent, and a compound to be delivered. In preferred embodiments, the compound to be delivered comprises nucleic acid. Also, certain preferred cell delivery systems include a targeting agent, preferably covalently linked to one or more of the endosomolytic agent, the delivery agent, and the delivery compound.
In one particularly preferred embodiment of the cell delivery system of the present invention, the endosomolytic agent comprises or consists of polyhistidine, the delivery agent comprises or consists of polylysine, and the delivery compound comprises or consists of nucleic acid, preferably DNA. The polyhistidine and polylysine may be mixed together as separate components or may be formulated together as a single linear or branched copolymer. That is, any appropriate combination of polyhistidine and polylysine, polyhistidine and lysine, or histidine and polylysine may be employed in accordance with the present invention.
DEFINITIONS
“Biocompatible”—The term “biocompatible”, as used herein is intended to describe compounds that are not toxic to cells. Compounds are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death and do not induce inflammation or other such adverse effects in vivo.
“Biodegradable”—As used herein, “biodegradable” compounds are those that, when introduced into cells, are broken down by the cellular machinery into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed).
“Biomolecules”—The term “biomolecules”, as used herein, refers to molecules (e.g., proteins, amino acids, nucleic acids, nucleotides, carbohydrates, sugars, lipids, etc.) that are found in living cells in nature.
“Known endosomolytic agents”—The phrase “known endosomolytic agents”, as used herein, refers to a particular set of compounds: chloroquine, fusogenic peptides, inactivated adenoviruses, and polyethyleneimine, that were known on the day the present application was filed to have osmolytic capabilities. The classification of such compounds as “known” is not intended to represent that such compounds constitute prior
Langer Robert S.
Pack Daniel W.
Putnam David A.
Baker C. Hunter
Choate Hall & Stewart
Jarrell Brenda H.
Lacourciere Karen
Massachusetts Institute of Technology
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