Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-05-04
2001-03-27
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C428S402200, C530S300000, C536S023100, C514S04400A
Reexamination Certificate
active
06207456
ABSTRACT:
FIELD OF THE INVENTION
The present invention is generally in the field of gene delivery and concerns a delivery vehicle for delivery of nucleic acid molecules to cells, both in vivo and ex vivo.
BACKGROUND OF THE INVENTION
The development of gene therapy for inherited or acquired diseases is dependent on the establishment of safe and efficient gene delivery systems. One method for in vivo gene transfer uses viruses to transfer the exogenous gene into cells, but this method also transfers viral genes into the cells which may produce undesirable effects. In addition, contamination by wild type viruses is also a risk in this method.
Another method for in vivo gene transfer uses a complex of positively charged liposomes (composed of synthetic lipids) that bind to the DNA (the DNA is not encapsulated in the liposomes) (Zhu, N., et al.,
Science,
261:209-211, 1993). However, the toxicity of positively charged liposomes (Raz, E., et al., In:
Vaccines
, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y., 1994) limits the use of this method.
A complex of poly-L-lysine coupled to DNA and a ligand which can be targeted to the cell surface was also used for in vivo gene transfer (Wu, G. Y. and Wu, C. H.,
J. Biol. Chem.,
263:14621-14624, 1988; Perales, J. C., et al.,
Proc. Natl. Acad. Sci.
, USA 91:4086-4090, 1994). However, transient expression of the transferred gene and immunogenicity of the complex limits the use of this method. Neutral or negatively charged liposomes (composed of synthetic or natural lipids) that encapsulate DNA were hitherto used for in vivo gene transfer (Nicolau, C., et al.,
Proc. Natl. Acad. Sci.
, USA, 80:1060-1072, 1983; Liebiger, B., et al.,
Biochem. Biophys. Res. Commun.,
174:1223-1231, 1991).
Such liposomes are non-toxic, non-immunogenic and biode-gradable (Storm et al. In: Gregoriadis, G. (Ed),
Liposome Technology,
2nd Ed. CRC Press, Baca Rator, Fla. 1993, 00.345-383) and are therefore good candidates for repetitive high dose treatment. However, the gene transfer efficiency in these experiments was low. One possible explanation for the low efficiency is that the DNA, which is encapsulated in liposomes and enters the cytoplasm through the lysosome (Cudd, A., and Nicolau, C.,
Biochim. Biophys. Acta,
845:477-491, 1985), is degraded by active DNases in the lysosome.
Therefore, treatments that inhibit lysosome activities can increase the amount of liposome-encapsulated DNA inside the cells.
Indeed, adenovirus particle and peptides derived from the N-terminal of the Influenza virus hemagglutinin subunit HA-2, which can disrupt membrane at low pH and therefore disrupt the lysosome membrane, were shown to enhance in vitro gene transfer efficiency when coupled to poly-L-lysine and DNA (Curiel, D. T., et al.,
Proc. Natl. Acad. Sci.
, USA, 88:8850-8854, 1993: Wagner, E., et al.,
Proc. Natl. Acad. Sci.
, USA, 89:7934-7938, 1992).
The efficiency of cationic-liposome mediated in vitro DNA transfection into cells was also shown to be increased when the cationic-liposomes were mixed with the DNA to be transfected together with two additional peptides derived from the Influenza virus hemagglutinin protein to form a complex between these three components (Kamata, H. et al. Nuclear Acid Research 22:536-537, 1994).
Fusion of liposomes prepared by reverse-phase evaporation (RPE) carrying DNA or protein molecules to target cells was shown to be mediated by Sendai virus proteins (Kameda, Y. et al,
Exp. Cell Res.
173:56, 1987) or influenza virus proteins (Lapidot, M., Loyter, A.,
Exp. Cell Res.
189:241-246, 1990; Tikchonenko, T. I. et al,
Gene
63:321-330, 1988) which were introduced into the liposome membrane (i,e, were present on the liposome's outer surface). The introduction of the DNA or protein encapsulated in the liposome was dependent on the presence of an active viral fusion protein and, therefore, either intact Sendai or influenza virus particles or their reconstituted envelopes were required. Liposomes containing modified reconstituted viral envelopes integrated in their lipid bilayer (Gould-Fogerite, S. et al,
Gene,
84, 429-438, 1989) were also prepared by the protein-cochleate technique. Such liposomes were used for stable gene transfer and expression in animals.
All the above mentioned liposomes have large viral particles or reconstituted viral envelopes integrated in their lipid membrane which protrude outwards from the liposome membrane. In vivo administration of liposomes carrying such large viral particles may be hindered by the high antigenicity of such large particles as well as occasionally by their toxic effects.
SUMMARY OF THE INVENTION
It is the object of the present invention to provide a novel polynucleic acid delivery system.
It is a further object of the invention to provide a method for the introduction of a polynucleic acid sequences into cells.
The invention concerns a novel composition for liposome-mediated ex vivo (particularly to cells removed from the body and returned to the body following a genetic manipulation) and in vivo transfer of nucleic acid sequences to cells. The formulation comprises liposomes in which a nucleic acid sequence is co-capsulated with a short peptide that disrupts membranes at a low pH.
The present invention thus provides a composition comprising:
i. a polynucleic acid molecule;
ii. a liposome; and
iii. a peptide comprised of less than about 50 amino acids which disrupts membranes under acidic conditions;
said peptide and the polynucleic acid molecule being encapsulated within the liposome.
The polynucleic acid molecule may be a DNA molecule, an RNA molecule, a moleclue consisting of both ribonucleotides and deoxy-nucleotides (RNA/DNA hybrid). The polynucleic acid molecule may be a large nucleic acid construct, such as a plasmid, an oligonucleotide, etc. The polynucleic acid is typically encapsulated within the aqueous interior of the liposome.
The liposomes used in accordance with the invention may be multilamellar or unilamellars. Most types of liposomes belong to either one of the following three types: multilamellar vesicles (MLV), small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV). MLVs typically form spontaneously upon hydration of dried phospholipids. SUVs, may be formed from MLVs by sonication and unlike the multilayered, onion skin-like structure of MLVs, they are single layered. SUVs are small with a high surface-to-volume ratio and thus have the lowest capture volume of aqueous space per weight of lipid.
As distinct from SUVs, LUVs have a large aqueous compartment and a single, or at times a few, lipid layers.
The liposomes may be comprised of a variety of lipids, including phospholipids, glycolipids, etc. Preferably phospholipids constitute a major component in the liposomes' membranes. Preferred phospholipids are &agr;-lecithines (also known as phosphatidyl-cholines), which are mixtures of diglyceride of stearic, palmitic and oleic acids linked to the choline ester of phosphoric acid. Lecithines are found in and obtainable from animals and plants. Preferred sources of lecithines are eggs, soybeans, animal tissues such as brain, heart, and the like. Lecithines can also be produced synthetically. As will no doubt be appreciated by the artisan, the source of the phospholipid is immaterial to the present invention and any phospholipid will likely be suitable.
Examples of specific phosphatides are L-&agr;-(distearoyl) lecithin, L-&agr;(diapalmitoyl) lecithin, L-&agr;-phosphatide acid, L-&agr;-(dilauroyl)-phosphatidic acid, L-&agr;-(dimyristoyl) phosphatidic acid, L-&agr;-(dioleoyl) phosphatidic acid, DL-&agr;-(dipalmitoyl) phosphatidic acid, L-&agr;-(distearoyl) phosphatidic acid, and the various types of L-&agr;-phosphatidylcholines prepared from brain, liver, egg yolk, heart, soybean and the like, or synthetically, and salts thereof. Other suitable modifications include the controlled peroxidation of the fatty acyl residue cross-linkers in the phosphatidylcholines (PC) and the zwitterionic amphiphates which form micelles by themselves or when mixed wit
Baru Moshe
Nur Israel
Brusca John S.
Oliff & Berridg,e PLC
Opperbas Holding B.V.
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