Follow us on Twitter
twitter icon@FreshPatents

Browse patents:
Next
Prev

High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them




Title: High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them.
Abstract: The present invention relates to novel fusogenic vesicles as highly efficient and versatile encapsulation systems for delivering a substance of choice, such as nucleic acids, proteins, peptides, antigens, pharmaceutical drugs and cosmetic agents to cells and tissues. ...


Browse recent Pevion Biotech Ltd. patents


USPTO Applicaton #: #20090280163
Inventors: Sonia Vadrucci, Joseph Brunner, Rinaldo Zubriggen


The Patent Description & Claims data below is from USPTO Patent Application 20090280163, High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/428,435, filed Nov. 21, 2002, the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

- Top of Page


The present invention relates to novel fusogenic vesicles as highly efficient and versatile encapsulation systems for delivering a substance of choice, such as nucleic acids, proteins, peptides, antigens, pharmaceutical drugs and cosmetic agents to cells and tissues.

BACKGROUND

- Top of Page


OF THE INVENTION

Various publications or patents are referred to in parentheses throughout this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein.

One of the paramount goals of medical therapy is the efficient delivery of therapeutic substances to the site of disease. While some therapeutic substances can be delivered in free form, others require a carrier or vector in order to reach and enter their final destination, either due to their rapid clearance from the area of introduction or their inability to cross biological barriers, or due to their systemic toxicity. Delivery of substances to cells and tissues requires vectors which are efficient, flexible, easy to prepare and safe. Currently available methods for delivering substances to eukaryotic cells involve the use of either viral or non-viral vectors. Viral vectors are replication-defective viruses with part of their coding sequences replaced by that of a therapeutic gene. Although recombinant viruses are highly efficient gene delivery and expression vectors, they are currently limited to the delivery of nucleic acids and their safety profiles have not yet been established for medical use in humans.

Most non-viral delivery systems operate at the following levels: loading of the delivery vector with a substance of interest (e.g. proteins, peptides, nucleic acids, pharmaceutical or other therapeutic drugs), endocytosis, and in the case of gene delivery, nuclear targeting and entry. The major drawback of non-viral systems, such as liposomes, is their low delivery efficiency to cells (Chu et al., id.; Legendre and Szoka, 1992, Pharmaceut. Res. Vol. 9, P. 1235), presumably due to the absence of fusion-mediating molecules on the surface of the liposomes. A hybrid type of delivery system, the virosome, combines the efficient delivery mechanism of viruses with the versatility and safety of non-viral delivery systems. Virosomes are reconstituted envelopes without the infectious nucleocapsids and the genetic material that can be derived from a variety of viruses. These virosomes are functional, in that their membrane fusion activity closely mimics the well-defined low-pH-dependent membrane fusion activity of the intact virus, which is solely mediated by the viral fusion protein. Like viruses, virosomes are rapidly internalized by receptor-mediated endocytosis. In contrast to viral systems virosomes are safe, since the infectious nucleocapsid of the virus has been removed. Thus, virosomes represent a promising carrier system for the delivery of a wide variety of different substances, either encapsulated in their aqueous interior or co-reconstituted in their membranes. Co-reconstitution of different receptors within the virosomal membrane, furthermore, allows the targeting of virosomes to different cells or tissues. So far, virosomes are mainly used as vaccines by adding antigen onto the surface of the virosomes.

A major limitation of the protocol currently used to prepare virosomes is that it does not result in high encapsulation efficiency. At the lipid concentration at which virosomes are produced (˜1 mM lipid), and given their mean diameter of approximately 200 nm, less than 1% of the aqueous phase will be entrapped within the virosomes (Schoen et al., J. Liposome Res. 3: 767-792, 1993). Such low entrapment rates reduce virosome-mediated efficiency of drug or gene delivery to cells. The development of new/novel, more efficiently loaded vesicles that retain the advantageous fusion properties of virosomes, as well as methods of making, loading, and delivering them would thus be a highly desirable goal in the field of therapeutic drug, protein and gene delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

- Top of Page


FIG. 1: This figure shows the simplified scheme representing the overall strategy of the invention. In a first fusion step, chimeric virosomes are fused at 4° C. (pH 5.0) with liposomes containing the substance to be delivered, (in this example DNA) mediated by X-31 HA. Subsequently, the resulting neutralized fusion products are incubated with cells. After receptor-mediated endocytosis, the virosome-like proteoliposomes now containing the substance to be delivered undergo a second round of fusion, triggered by the low pH within endosomes and mediated by PR8/34 HA, releasing the entrapped substance into the cytosol.

FIG. 2: This figure shows the presence of both types of fusion proteins in the membrane of the chimeric virosomes. SDS-PAGE of X-31 and PR8 virus and virosomes. Approx. 10 μg virus and 3 μg virosomes were loaded under non-reducing conditions on a SDS-Tris/Tricine 10% (v/v) polyacrylamide gel (Schäigger et al., Anal. Biochem. 166: 368-379, 1987). The polyacrylamide gel was stained with Coomassie blue. Lane 1: X-31 virus. Lane 2: X-31 virosomes. Lane 3: chimeric virosomes. Lane 4: PR8 virosomes. Lane 5: PR8 virus.

FIG. 3: This figure shows the morphology of the chimeric virosomes by cryo-TEM. They are unilamellar vesicles with diameters ranging from 150-250 nm. Scale bar 100 nm.

FIG. 4: This figure shows the fusion activity of X-31, PR8 viruses, virosomes and X-31/PR8 chimeric virosomes with liposomes. Fluorescence was recorded continuously at excitation and emission wavelengths of 465 nm and 530 nm, respectively. Measurements were carried out with a SPEX-Fluorolog-2 fluorometer (SPEX Industries, Inc., Edison, N.J., USA) equipped with a thermostated cuvette holder and a magnetic stirring device. For calibration of the fluorescence scale the initial residual fluorescence of the liposomes was set to zero and the fluorescence at infinite probe dilution to 100%. The latter value was determined by addition of Triton X-100 (0.5% v/v). 1) X-31 virus and virosomes fused with POPC/POPG-LUVs. 2) PR8/34 virus and virosomes incubated with POPC/POPG-LUVs. 3) Chimeric virosomes incubated with POPC/POPG-LUVs. a) fusion measurements at 4° C.; b) Virus and virosomes were incubated at 4° C. and pH 5.0 for 1 h with POPC/DDAB-LUVs before fusion measurements at 37° C.

FIG. 5: This figure shows the encapsulation efficiency of DNA into POPC/DDAB-liposomes. DNA-containing liposomes were prepared by the freeze/thaw method as described in the examples and digested for 3 h at 37° C. with DNase I/Exonuclease III. After that, encapsulated DNA was phenol/chloroform extracted and subjected to gel electrophoresis on a 1% agarose gel. In lane M 1 Kb plus Ladder marker was loaded. As control 100 ng (lane 1) and 200 ng plasmid-DNA (lane 2) were directly loaded on the gel. As another control untreated (not DNase I digested) DNA-liposomes corresponding to 20 ng (lane 3), 50 ng (lane 4), 100 ng (lane 5) and 200 ng (lane 6) plasmid were phenol/chloroform extracted. The aqueous phase was loaded on the gel. DNase I digested liposomes corresponding to an initial DNA-amount of 50 ng (lane 7) and 100 ng (lane 8) were treated like the samples before. Lane 9 and 10 correspond to the same amount of liposomes, except that liposomes had been solubilized with 1% (v/v) Triton X-100 before DNase I treatment. In lane 11 and 12 DNA was not encapsulated, but added to empty liposomes. Also these liposomes were treated as described above.

FIG. 6: This figure shows the morphology of loaded liposomes by cryo-TEM of. They are unilamellar and measure between 100-150 nm in diameter. Scale bar 100 nm.

FIG. 7: This figure shows the products of the fusion between the chimeric virosomes and the loaded liposomes by cryo-TEM before (upper panels) and after extrusion (lower panels). Before extrusion, the vesicles are of widely varying sizes with some vesicles exceeding 900 nm. This result indicates multiple rounds of fusion. Scale bars upper panels: 500 nm; scale bars lower panels: left: 100 nm; right: 200 nm.

FIG. 8: This figure demonstrates the fusogenicity of the extruded vesicles resulting from the fusion of chimeric virosomes with liposomes. Remarkably, the vesicles retain their fusion activity. The proteoliposomes were extruded through nucleopore membranes. Fluorescence was recorded continuously at excitation and emission wavelengths of 465 nm and 530 nm, respectively. Measurements were carried out with a SPEX-Fluorolog-2 fluorometer equipped with a thermostated cuvette holder and a magnetic stirring device. 100% corresponded to infinite probe dilution, determined after addition of Triton X-100 (0.5% v/v). Fusion measurements were carried out at 37° C. and pH 5.0 with POPC/POPG (4:1)-LUVs containing 0.6% N-NBD-PE and N-Rh-PE.

FIG. 9: This figure shows that the proteoliposomes are rapidly internalized by cells. Rhodamine-labeled proteoliposomes are detected in MDCK and HeLa cells. Fused virosomes were bound at 4° C. for 1 h to HeLa and MDCK cells. After that, unbound material was washed away. Cells were immediately fixed (A), or incubated for another 15 min at 37° C., fixed with 3.7% (v/v) formaldehyde and analyzed by fluorescence microscopy (B).

FIG. 10: This figure shows the rapid uptake of proteoliposomes loaded with Texas Red dextran by HeLa cells. HeLa cells were incubated with Texas Red dextran-containing proteoliposomes for 1 h at 4° C. After that, unbound material was washed away and fixed with 3.7% (v/v) formaldehyde (A). Otherwise, cells were further incubated for 15 min (B), 2 h (C) or 5 h (D) at 37° C. After fixation, cells were analyzed by fluorescence microscopy. Objective magnification: for (A) 63×; for (B), (C), (D) 100×.

DETAILED DESCRIPTION

- Top of Page


OF THE INVENTION

One of the problems in current virosome technology is the lack of methods for the efficient entrapment of a solute, e.g. protein, nucleic acid, or pharmaceutical drug. The present invention provides a novel approach that circumvents the problems of low virosomal entrapment efficiencies.

Therefore the invention provides a fusogenic vesicle, composed of virosomal and liposomal lipids that is capable of encapsulating at least one therapeutic or immunologically active substance whereby said fusogenic vesicle is comprised of fusion proteins, preferably at least two different fusion proteins or peptides with distinct fusion characteristics. Preferably, the vesicle is unilamellar. The vesicle has a diameter generally in the range of 100 to 600 nm, and preferably a diameter of between 100 nm and 300 nm or a diameter of between 200 nm and 400 nm, depending on the specific vesicle.

The invention takes advantage of the fact that liposomes, which can be prepared at very high lipid concentrations, have high encapsulation efficiencies. Thus, the invention provides a methodology that combines the high loading capacity of liposomes with the efficient cell-fusion and delivery of virosomes, resulting in substantially increased entrapment of solutes, like proteins, nucleic acids, and pharmaceutical drugs, within functional chimeric virosomes of controlled size that are capable of efficiently delivering therapeutic or immunologically active substances to cells.

Accordingly, in a preferred embodiment of the invention, the substance of interest is first loaded into, or encapsulated by, liposomes. The liposomal lipids which can be used in the present invention include cationic lipids, synthetic lipids, glycolipids, phospholipids cholesterol or derivatives thereof, and equivalent molecules known to those of skill in the art. Phospholipids can comprise preferably phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol with varying fatty acyl compositions. Cationic lipids can comprise preferably DOTMA (N-[(1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, DODAC (N,N-dioleyl-N,N,-dimethylammonium chloride), DDAB (didodecyldimethylammonium bromide) and stearylamine or other aliphatic amines and the like. They are generally formulated as small unilamellar liposomes in a mixture with DOPE (dioleoylphosphatidyl ethanolamine) that is widely used as a ‘helper’ lipid to facilitate disruption of the endosomal membrane. Most preferably the liposomal lipids of the liposomes comprise POPC/DDAB.

In a second step, liposomes containing the substance of interest will be fused with chimeric virosomes having different types of fusion proteins in their membranes. The term “fusion protein” refers to peptides or proteins capable of inducing and/or promoting a fusion reaction between the fusogenic vesicle membrane and a biological membrane of the target cell. After resizing, the products of the liposome-virosome fusion will be virosome-like particles, or proteoliposomes, that contain the substance of interest in their internal cavity and are still capable of undergoing a second fusion step, under different conditions, with biological membranes in order to deliver the therapeutic or immunologically active substance.

The fusogenic vesicle of the present invention is capable of encapsulating at least one substance, preferably a therapeutic or an immunologically active substance. Examples of preferred substances suitable for encapsulation into the fusogenic vesicles are DNA, RNA, siRNA, proteins, peptides, amino acids and pharmaceutical active substances or derivatives thereof. Preferably the at least one substance is a pharmaceutical drug, an antigen, a cosmetic agent or a mixture thereof. The substance can also be a precursor to said pharmaceutical drug, antigen, or cosmetic agent that is converted into the final agent by the cell, tissue or interstitium, or by some other mechanism.

Examples of cosmetic substances are well known in the art and may comprise antipsoriatics or antifungals for dermatological use. Examples of therapeutic substances or pharmaceutical drugs are also well known in the art and may comprise anaesthetics, angiogenesis inhibitors, anti-acne preparations, anti-allergica, antibiotics, chemotherapeutics, antihistamines, antiinflammatory or antiinfective agents, antineoplastic agents, antigens, antiprotozoals, antirheumatic agents, antiviral vaccines, antiviral agents, aptoptosis inducing agents, bacterial vaccines, chemotherapeutics, cytostatica, immunosuppressive agents, laxatives, psycholeptics.

Preferred examples of cosmetic agents include tars and nystatin, while preferred examples of pharmaceutical drugs or immuno-active substances include doxorubicin, vinblastine, cisplatin, methotrexate, cyclosporine, ibuprofen, HCV-based T-cell antigens, and tumor-specific and tumor-associated antigens.

Encapsulation of the therapeutic substances, such as proteins, peptides, nucleic acids, pharmaceutical, chemotherapeutic or cosmetic drugs into liposomes can be performed by any method known in the art, including the procedures described in Monnard et al., Biochim. Biophys. Acta 1329: 39-50; 1997, in Wagner et al., J. Liposome Res., 12(3) 271-283, 2002, or in Oberholzer et al., Biochim. Biophys. Acta 1416: 57-68; 1999, among many other well-known methods. In a preferred embodiment of the invention, high liposomal encapsulation efficiencies are achieved by the freeze/thaw technique used to prepare pure lipid vesicles. With this method, approximately 50% of the initial amount of a linearized plasmid over 3 kb can be entrapped within large unilamellar vesicles (LUVs) consisting preferably of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/didodecyldimethylammonium bromide (DDAB) (Monnard et al., Biochim. Biophys. Acta 1329: 39-50, 1997).




← Previous       Next →
Advertise on FreshPatents.com - Rates & Info


You can also Monitor Keywords and Search for tracking patents relating to this High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them patent application.

###


Browse recent Pevion Biotech Ltd. patents

Keyword Monitor How KEYWORD MONITOR works... a FREE service from FreshPatents
1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored.
3. Each week you receive an email with patent applications related to your keywords.  
Start now! - Receive info on patent apps like High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them or other areas of interest.
###


Previous Patent Application:
High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them
Next Patent Application:
Therapy for human cancers using cisplatin and other drugs or genes encapsulated into liposomes
Industry Class:
Drug, bio-affecting and body treating compositions
Thank you for viewing the High-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them patent info.
- - -

Results in 0.10898 seconds


Other interesting Freshpatents.com categories:
Amazon , Microsoft , Boeing , IBM , Facebook

###

Data source: patent applications published in the public domain by the United States Patent and Trademark Office (USPTO). Information published here is for research/educational purposes only. FreshPatents is not affiliated with the USPTO, assignee companies, inventors, law firms or other assignees. Patent applications, documents and images may contain trademarks of the respective companies/authors. FreshPatents is not responsible for the accuracy, validity or otherwise contents of these public document patent application filings. When possible a complete PDF is provided, however, in some cases the presented document/images is an abstract or sampling of the full patent application for display purposes. FreshPatents.com Terms/Support
-g2-0.274

66.232.115.224
Browse patents:
Next
Prev

stats Patent Info
Application #
US 20090280163 A1
Publish Date
11/12/2009
Document #
File Date
12/31/1969
USPTO Class
Other USPTO Classes
International Class
/
Drawings
0


Cosmetic Agents Fusogenic Vesicle Vesicle Vesicles

Follow us on Twitter
twitter icon@FreshPatents

Pevion Biotech Ltd.


Browse recent Pevion Biotech Ltd. patents



Drug, Bio-affecting And Body Treating Compositions   Preparations Characterized By Special Physical Form   Liposomes  

Browse patents:
Next
Prev
20091112|20090280163|high-efficiency fusogenic vesicles, methods of producing them, and pharmaceutical compositions containing them|The present invention relates to novel fusogenic vesicles as highly efficient and versatile encapsulation systems for delivering a substance of choice, such as nucleic acids, proteins, peptides, antigens, pharmaceutical drugs and cosmetic agents to cells and tissues. |Pevion-Biotech-Ltd
';