Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-12-14
2002-01-22
Priebe, Scott D. (Department: 1633)
Chemistry: molecular biology and microbiology
Vector, per se
C435S458000, C435S440000, C424S450000, C514S04400A, C530S350000, C536S023400, C536S023500
Reexamination Certificate
active
06340591
ABSTRACT:
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to a lipidic vector system comprising a multi-layered lipid bilayer structure called a cochleate precipitate. The layers of the lipid bilayer structure are ionically bound together by a cation.
One or more therapeutic nucleotide sequences coding for a therapeutically beneficial molecule and one or more proteins that facilitate integration of the therapeutic nucleotide sequences physically associated with the cochleate precipitate.
The proteins are preferably adeno-associated virus (AAV) Rep 68 and Rep 78. The therapeutic nucleotide sequence is preferably positioned between AAV inverted terminal repeats (ITRs).
Upon contact with a lipid bilayer of a target cell, the cochleate vector structure delivers one or more of the therapeutic nucleotide sequences and one or more of the proteins to the interior of the target cell. Upon entry into the cell the one or more proteins facilitate the integration of the therapeutic nucleotide sequence(s) into the genome of the host cell.
BACKGROUND OF THE INVENTION
Recent advances in molecular biology have increased the scientific understanding of the genetic basis for disease and have provided the tools for novel advances in gene therapy. For example, it is now possible to produce genetically engineered nucleotide sequences capable of expressing therapeutic molecules. Yet, major obstacles have remained, and one such obstacle has been the lack of effective means for delivering these therapeutic nucleotide sequences to the interior of a target cell in a form capable of integrating into the target cell's genome. This invention relates to a vector delivery system capable of delivering genetic materials to the interior of a cell along with the molecules necessary for the integration of such genetic materials into the genome of the target cell.
The vector delivery system of the present invention comprises a lipidic structure called a cochleate precipitate or, simply, a cochleate. The cochleate comprises a multi-layered lipid bilayer structure. The multi-layered lipid bilayer structure generally comprises a membrane phospholipid containing a negatively charged head group and a cation such as calcium (Ca
++
). The cation serves as a bridge, ionically binding to the negatively-charged head groups of the phospholipid groups and thus linking together the individual lipid bilayers of the limit-layered structure.
Cochleates consist of alternating sheets of cation-complexed lipid. In a preferred mode of the invention, the multi-layered lipid bilayer structure exists as a continuous lipid bilayer sheet rolled up into a spiral conformation. The cation maintains the cochleate structure by ionically binding to the negatively charged head groups in the opposing lipid bilayers. For example, where the cation is C
++
, one positive charge of the Ca
++
attracts a negatively charged phospholipid headgroup in one bilayer, and the other positive charge attracts a phospholipid headgroup in the opposing bilayer.
Cochleates are highly stable and can be stored in calcium-containing buffer. Cochleates can also be lyophilized to a powder, stored at room temperature, and reconstituted with liquid prior to administration.
While other lipidic vector delivery systems are known (See Lee et. al., “Lipidic Vector Systems for Gene Transfer”, Critical Reviews in Drug, Carrier Systems 14(2): 173-206 (1997)), they are typically in the form of liposomes and arc substantially different from the cochleate vector delivery system described herein. A liposome is a fluid-filled compartment bounded by a fluid lipid bilayer. Materials, such as DNA or protein, can be contained within a liposome, and such materials can be delivered to the interior of a cell by endocytic uptake or, in special instances, fusion of the liposome with the cell membrane.
Unlike cochleate structures, harsh environmental conditions, such as extreme pH levels or the presence of lipid degrading enzymes, render the lipid bilayer of a liposome susceptible to instability and compromise the membrane barrier. Compromise of the membrane barrier renders the contents of the liposome subject to attack by external elements. For example, degradative enzymes, such as proteases and nucleases can degrade proteins and polypeptides within the compromised liposome.
An additional difference between liposomes and cochleate is the presence of divalent cations. Cochleates are prepared by calcium induced fusion of liposomes. Cochleates can contain, for example, one-half the molar concentration of divalent cations relative to the molar concentration of phospholipids. The divalent cations organize the negatively charged lipid bilayers into solid sheets that roll or stack upon themselves, excluding water.
Cochleates are multi-layered, highly stable structures composed of non-toxic and non-inflammatory natural products. They are solid, lyophilizable precipitates containing little or no internal aqueous space. Whereas dehydration of liposomes, e.g., by lyophilization, destroys the morphology and integrity of liposomes, such dehydration has no adverse effects on cochleate morphology or functions. The layers of the cochleate are composed of alternating sheets of negatively charged phospholipid and calcium. This unique structure protects associated, “encochleated,” molecules from degradation. Since the entire cochleate structure is a series of solid layers, components within the interior of the cochleate structure remain intact even though the outer layers of the cochleate may be exposed to harsh environmental conditions or enzymes.
The formulation of integrative DNA protein complexes or the use of these complexes as gene transfer vehicles has not heretofore been described.
Calcium induced perturbations of membranes containing negatively charged lipids, and the subsequent, resulting membrane fusion events, are important mechanisms in many natural membrane fusion processes. Accordingly, while the definitive mechanism of cochleate delivery is unknown, it is hypothesized that cochleates act as membrane fusion intermediates. According to this theory, as the calcium rich membrane of a cochleate approaches a natural membrane, a perturbation and reordering of the cell membrane is induced, resulting in a fusion event between the outer layer of the cochleate and the cell membrane. This fusion results in the delivery of a small amount of the encochleated material into the cytoplasm of the cell. Theoretically, the cochleate can then break free of the cell and be available for another fusion event, either with this or another cell. Alternatively, the cochleate may be taken up by endocytosis, and fuse with the cellular membranes from within. In contrast, the lipid bilayer of most liposomes is highly thermodynamically stable and resists fusion with other liposomes or with other membrane bound structures.
The membrane fusion hypothesis is consistent with the observation that many naturally occurring membrane fusion events involve the interaction of calcium with negatively charged phospholipids, (generally phosphatidylserine and phosphatidylglycerol). This hypothesis is also consistent with experimental studies. For example, the ability of cochleates to mediate the induction of antigen specific, CD8
+
cytotoxic lymphocytes supports the hypothesis that cochleates facilitate the cytoplasmic delivery of cochleate-associated macromolecules. And, immunological studies indicating a slow, long-term presentation of antigen are consistent with the theory that a single cochleate undergoes multiple fusion events over an extended period of time.
The present invention makes use of cochleates as delivery vehicles for genetic materials and proteins that facilitate integration of the genetic materials into the host genome. In one example, the genetic materials and proteins are from the adeno-associated virus (AAV). AAV is a naturally defective, single stranded DNA parvovirus that is commonly used as a vector.
Wild-type AAV generally requires co-infection with a helper virus in or
Gould-Fogerite Susan
Mannino Raphael James
Margolis David
Priebe Scott D.
Sorbello Eleanor
Sughrue & Mion, PLLC
University of Maryland
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