US20050079212A1 - Methods for encapsulating plasmids in lipid bilayers - Google Patents

Methods for encapsulating plasmids in lipid bilayers Download PDF

Info

Publication number
US20050079212A1
US20050079212A1 US10/956,425 US95642504A US2005079212A1 US 20050079212 A1 US20050079212 A1 US 20050079212A1 US 95642504 A US95642504 A US 95642504A US 2005079212 A1 US2005079212 A1 US 2005079212A1
Authority
US
United States
Prior art keywords
plasmid
lipid
method
particles
peg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/956,425
Inventor
Jeffery Wheeler
Michael Hope
Pieter Cullis
Marcel Bally
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of British Columbia
Arbutus Biopharma Corp
Original Assignee
Inex Pharmaceuticals Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
Priority to US08/484,282 priority Critical patent/US5981501A/en
Priority to US09/436,933 priority patent/US6534484B1/en
Priority to US10/374,673 priority patent/US6815432B2/en
Application filed by Inex Pharmaceuticals Corp filed Critical Inex Pharmaceuticals Corp
Priority to US10/956,425 priority patent/US20050079212A1/en
Publication of US20050079212A1 publication Critical patent/US20050079212A1/en
Assigned to THE UNIVERSITY OF BRITISH COLUMBIA reassignment THE UNIVERSITY OF BRITISH COLUMBIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: INEX PHARMACEUTICALS CORPORATION
Assigned to THE UNIVERSITY OF BRITISH COLUMBIA, TEKMIRA PHARMACEUTICALS CORPORATION reassignment THE UNIVERSITY OF BRITISH COLUMBIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: INEX PHARMACEUTICALS CORPORATION
Assigned to SILICON VALLEY BANK reassignment SILICON VALLEY BANK SECURITY AGREEMENT Assignors: TEKMIRA PHARMACEUTICALS CORPORATION
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=23923500&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20050079212(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application status is Abandoned legal-status Critical

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • A61K9/1271Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00Chemistry: analytical and immunological testing
    • Y10S436/829Liposomes, e.g. encapsulation
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S514/00Drug, bio-affecting and body treating compositions
    • Y10S514/851Cystic fibrosis
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00Nanotechnology
    • Y10S977/70Nanostructure
    • Y10S977/788Of specified organic or carbon-based composition
    • Y10S977/797Lipid particle
    • Y10S977/798Lipid particle having internalized material
    • Y10S977/799Containing biological material
    • Y10S977/80Nucleic acid, e.g. DNA or RNA
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00Nanotechnology
    • Y10S977/902Specified use of nanostructure
    • Y10S977/904Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
    • Y10S977/906Drug delivery
    • Y10S977/907Liposome

Abstract

Plasmid-lipid particles which are useful for transfection of cells in vitro or in vivo are described. The particles can be formed using either detergent dialysis methods or methods which utilize organic solvents. The particles are typically 65-85 nm, fully encapsulate the plasmid and are serum-stable.

Description

    FIELD OF THE INVENTION
  • This invention relates to formulations for therapeutic nucleic acid delivery and methods for their preparation, and in particular to lipid encapsulated plasmids or antisense constructs. The invention provides a circulation-stable, characterizable delivery vehicle for the introduction of plasmids or antisense compounds into cells. These vehicles are safe, stable, and practical for clinical use.
  • BACKGROUND OF THE INVENTION
  • Gene therapy is an area of current interest which involves the introduction of genetic material into a cell to facilitate expression of a deficient protein. There are currently five major methods by which this is accomplished, namely: (i) calcium phosphate precipitation, (ii) DEAE-dextran complexes, (iii) electroporation, (iv) cationic lipid complexes and (v) reconstituted viruses or virosomes (see Chang, et al., Focus 10:88 (1988)). Cationic lipid complexes are presently the most effective generally used means of effecting transfection.
  • A number of different formulations incorporating cationic lipids are commercially available, namely (i) LIPOFECTIN® (which uses 1,2-dioleyloxy-3-(N,N,N-trimethyfamino)propane chloride, or DOTMA, see Eppstein, et al., U.S. Pat. No. 4,897,355); LIPOFECTAMINE® (which uses DOSPA, see Hawley-Nelson, et al., Focus 15(3):73 (1993)); and LIPOFECTACE® (which uses N,N-distearyl-N,N-dimethyl-ammonium bromide, or DDAB, see Rose, U.S. Pat. No. 5,279,833). Others have reported alternative cationic lipids that work in essentially the same manner but with different efficiencies, for example 1,2-dioleoyloxy-3-(N,N,N-trimethyamino)propane chloride, or DOTAP, see Stomatatos, et al., Biochemnistry 27:3917-3925 (1988)); glycerol based lipids (see Leventis, et al., Biochem. Biophys. Acta 1023:124 (1990); lipopolyamines (see, Behr, et al., U.S. Pat. No. 5,171,678) and cholesterol based lipids (see Epand, et al., WO 93/05162, and U.S. Pat. No. 5,283,185).
  • Others have noted that DOTMA and related compounds are significantly more active in transfection assays than their saturated analogues (see, Felgner, et al., WO91/16024). However, both DOTMA and DOSPA based formulations, despite being the most efficient of the cationic lipids in effecting transfection, are prohibitively expensive. DDAB on the other hand is inexpensive and readily available from chemical suppliers but is less effective than DOTMA in most cell lines. Another disadvantage of the current lipid systems is that they are not appropriate for intravenous injection.
  • An examination of the relationship between the chemical structure of the carrier vehicle and its efficiency of transfection has indicated that the characteristics which provide for effective transfection would make a carrier unstable in circulation (see, Ballas, et al., Biochim. Biophys. Acta 939:8-18 (1988)). Additionally, degradation either outside or inside the target cell retains a problem (see, Duzghines, Subcellular Biochemistry 11:195-286 (1985)). Others who have attempted to encapsulate DNA (Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); and Deamer, U.S. Pat. No. 4,515,736) made no efforts to ensure a safe, injectable formulation, or arrived at inefficient loading (Legendre, Pharm. Res. 9:1235-1242 (1992)).
  • Ideally, a delivery vehicle for a nucleic acid or plasmid will have the following characteristics: a) small enough and long lived enough to distribute from local injection sites when given intravenously, b) capable of carrying a large amount of DNA per particle to enable transfection of all sizes of genes and reduce the volume of injection, c) homogenous, d) reproducible, e) protective of DNA from extracellular degradation and f) capable of transfecting target cells in such a way that the DNA is not digested intracellularly.
  • The present invention provides such compositions and methods for their preparation and use.
  • SUMMARY OF THE INVENTION
  • In one aspect, the present invention provides methods for the preparation of serum-stable plasmid-lipid particles. In one group of these methods, a plasmid is combined with cationic lipids in a detergent solution to provide a coated plasmid-lipid complex. The complex is then contacted with non-cationic lipids to provide a solution of detergent, a plasmid-lipid complex and non-cationic lipids, and the detergent is then removed to provide a solution of serum-stable plasmid-lipid particles, in which the plasmid is encapsulated in a lipid bilayer. The particles, thus formed, have a size of about 50-150 nm.
  • In a related group of methods the serum-stable plasmid-lipid particles are formed by preparing a mixture of cationic lipids and non-cationic lipids in an organic solvent; contacting an aqueous solution of plasmid with the mixture of cationic and non-cationic lipids to provide a clear single phase; and removing the organic solvent to provide a suspension of plasmid-lipid particles, in which the plasmid is encapsulated in a lipid bilayer, and the particles are stable in serum and have a size of about 50-150 nm.
  • In another aspect, the present invention provides plasmid-lipid particles prepared by the above methods.
  • In yet another aspect, the present invention provides methods of transfecting cells using these plasmid-lipid particles.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 illustrates a liposome-mediated transfection using “sandwich-type” complexes of DNA.
  • FIG. 2 illustrates an aggregation and precipitation which commonly occurs during the entrapment of large nucleic acids in lipid complexes.
  • FIG. 3 provides a schematic representation of the preparation of plasmid-lipid particles using the methods of the present invention.
  • FIG. 4 illustrates the recovery of 3H-DNA from encapsulated particles following the reverse-phase preparation of the particles and extrusion through a 400 nm filter and a 200 nm filter. Lipid composition is POPC:DODAC:PEG-Cer(C20) in proportions as shown in Table 1.
  • FIG. 5 illustrates the recovery of 3H-DNA from particles prepared using a reverse-phase procedure. The particles were extruded through a 200 nm filter and eluted on a DEAE Sepharose CL-6B anion exchange column. The percent recovery reported is based on the amount recovered after filtration. Lipid composition is as in FIG. 4.
  • FIG. 6 illustrates the recovery of 14C-lipid from encapsulated particles following the reverse-phase preparation of the particles and extrusion through a 400 nm filter and a 200 nm filter. Lipid composition is as in FIG. 4.
  • FIG. 7 illustrates the recovery of 14C-lipid from particles prepared using a reverse-phase procedure. The particles were extruded through a 200 nm filter and eluted on a DEAE Sepharose CL-6B anion exchange column. The percent recovery reported is based on the amount recovered after filtration. Lipid composition is as in FIG. 4.
  • FIG. 8 illustrates recovery of 3H-DNA and 14C-lipids from particles prepared by detergent dialysis after elution on a DEAE Sepharose CL-6B anion exchange column in HBS, pH 7.4. Lipid composition is POPC:DODAC:PEG-Cer(C20) in proportions as shown in Table 2.
  • FIG. 9 illustrates recovery of 3H-DNA and 14C-lipids from particles prepared by detergent dialysis after elution on a DEAE Sepharose CL-6B anion exchange column in HBS, pH 7.4. Lipid composition is DOPE:DODAC:PEG-Cer(C20) in proportions as shown in Table 3.
  • FIG. 10 provides an elution profile of free 3H-DNA (pCMV4-CAT) on a Sepharose CL4B column in HBS, pH 7.4.
  • FIG. 11 provides an elution profile of free 3H-DNA (pCMV4-CAI) on a Sepharose CL-4B column in HBS, pH 7.4, after incubation in 80% mouse serum for 30 min at 37° C.
  • FIG. 12 shows the recovery of 3H-DNA and 14C-lipids from particles (prepared by reverse-phase methods) after incubation in 80% mouse serum for 15 min at 37° C. Lipid composition is POPC:DODAC:PEG-Cer(C20).
  • FIG. 13 shows the recovery of 3H-DNA and 14C-lipids from particles (prepared by detergent dialysis methods) after incubation in 80% mouse serum for 30 min at 37° C. Lipid composition is DOPE:DODAC:PEG-Cer(C20).
  • FIG. 14 provides a density gradient profile of 14C-lipid complexes prepared in the absence of DNA by reverse phase methods. Lipid composition is POPC:DODAC:PEG-Cer(C20).
  • FIG. 15 provides a density gradient profile of free 3H-DNA (pCMV4-CAT).
  • FIG. 16 provides a density gradient profile of 3H-DNA and 14C-lipid from particles prepared by reverse-phase methods. Lipid composition is as in FIG. 14.
  • FIG. 17 provides a density gradient profile of free 3H-DNA, 14C-lipid complexes prepared in the absence of DNA by detergent dialysis methods and 3H-DNA and 14C-lipid from DNA-lipid complexes prepared by detergent dialysis. Lipid composition is DOPE:DODAC:PEG-Cer(C20).
  • FIG. 18 provide a size distribution of DNA-lipid particles prepared by detergent dialysis methods. Lipid composition is DOPE:DODAC:PEG-Cer(C20).
  • FIG. 19 shows the clearance of 3H-DNA and 14C-lipid from particles (prepared by reverse-phase methods) after injection into IRC mice. The figure includes free 3H-DNA after injection as a comparison. Lipid composition is POPC:DODAC:PEG-Cer(C20).
  • FIG. 20 shows the clearance of 3H-DNA and 14C-lipid from particles (prepared by detergent dialysis methods) after injection into IRC mice. Lipid composition is DOPE:DODAC:PEG-Cer(C20)(83.5:6.5:10 mole %).
  • FIG. 21 shows the clearance of 3H-DNA and 14C-lipid from particles (prepared by detergent dialysis methods) after injection into IRC mice. Lipid composition is as in FIG. 20 except that PEG-Cer(C20) is replaced with PEG-Cer(C14).
  • FIG. 22 shows the results of in vivo transfection which occurs in the lungs of mice. Lipid composition is DOPE:DODAC:PEG-Cer(C20 or C14) (83.5:6.5:10 mole %).
  • FIG. 23 shows the results of in vivo transfection which occurs in the liver of mice. Lipid composition is as in FIG. 22.
  • FIG. 24 shows the results of in vivo transfection which occurs in the spleen of mice. Lipid composition is as in FIG. 22.
  • DETAILED DESCRIPTION OF THE INVENTION Contents
  • I. Glossary
    II. General
    III. Methods of Forming Plasmid-Lipid Particles
    IV. Pharmaceutical Preparations
    V. Administration of Plasmid-Lipid Particle Formulations
    VI. Examples
    VII. Conclusion

    I. Glossary
  • The following abbreviations are used herein: DC-Chol, 3β-(N-(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (see, Gao, et al., Biochem. Biophys. Res. Comm. 179:280-285 (1991)); DDAB, N,N-distearyl-N,N-dimethylammonium bromide; DMRIE, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide; DODAC, N,N-dioleyl-N,N-dimethylammonium chloride (see commonly owned patent application U.S. Ser. No. 08/316,399, incorporated herein by reference); DOGS, diheptadecylamidoglycyl spermidine; DOPE, 1,2-sn-dioleoylphoshatidyethanolamine; DOSPA, N-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate; DOTAP, N-(1-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOTMA, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; EPC, egg phosphatidylcholine; RT, room temperature; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HBS, HEPES buffered saline (150 mM NaCl and 20 mM HEPES); PEG-Cer-C20, 1-O-(2′-(ω-methoxypolyethyleneglycol)succinoyl)-2-N-arachidoyl-sphingosine; PEG-Cer-C14, 1-O-(2′-(ω-methoxypolyethyleneglycol)succinoyl)-2-N-myristoyl-sphingosine; PBS, phosphate-buffered saline; EGTA, ethylenebis(oxyethylenenitrilo)-tetraacetic acid; OGP, noctyl β-D-glycopyranoside (Sigma Chemical Co., St. Louis, Mo.); POPC, palmitoyl oleoyl phosphatidylcholine (Northern Lipids, Vancouver, BC); QELS, quasielastic light scattering; TBE, 89 mM Trisborate with 2 mM EDTA; and EDTA, Ethylenediaminetetraacetic acid (Fisher Scientific, Fair Lawn, N.J.);
  • The term “acyl” refers to a radical produced from an organic acid by removal of the hydroxyl group. Examples of acyl radicals include acetyl, pentanoyl, palmitoyl, stearoyl, myristoyl, caproyl and oleoyl.
  • The term “lipid” refers to any fatty acid derivative which is capable of forming a bilayer such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and other like groups. Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s). Preferred lipids are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine could be used. Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.
  • The term “non-cationic lipid” refers to any of a number of lipid species which exist either in an uncharged form, a neutral zwitterionic form, or an anionic form at physiological pH. Such lipids include, for example diacylphosphatidylcholine, diacylphosphatidylethanolamrine, cerarnide, sphingomyelin, cephalin, cardiolipin, and cerebrosides.
  • The term “cationic lipid” refers to any of a number of lipid species which carry a net positive charge at physiological pH. Such lipids include, but are not limited to, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECAMINE® (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic liposomes comprising DOGS from Promega Corp., Madison, Wis., USA).
  • The terms “transfection” and “transformation” are used herein interchangeably, and refer to the introduction of polyanionic materials, particularly nucleic acids, into cells. The term “lipofection” refers to the introduction of such materials using liposome or lipid-based complexes. The polyanionic materials can be in the form of DNA or RNA which is linked to expression vectors to facilitate gene expression after entry into the cell. Thus the polyanionic material used in the present invention is meant to include DNA having coding sequences for structural proteins, receptors and hormones, as well as transcriptional and translational regulatory elements (i.e., promoters, enhancers, terminators and signal sequences) and vectors. Methods of incorporating particular nucleic acids into expression vectors are well known to those of skill in the art, but are described in detail in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York (1987), both of which are incorporated herein by reference.
  • “Expression vectors”, “cloning vectors”, or “vectors” are often plasmids or other nucleic acid molecules that are able to replicate in a chosen host cell. Expression vectors may replicate autonomously, or they may replicate by being inserted into the genome of the host cell, by methods well known in the art. Vectors that replicate autonomously will have an origin of replication or autonomous replicating sequence (ARS) that is functional in the chosen host cell(s). Often, it is desirable for a vector to be usable in more than one host cell, e.g., in E. coli for cloning and construction, and in a mammalian cell for expression.
  • II. General
  • Although directed to the transfer of nucleic acids, and in particular to the transfer of plasmids to cells, the particles of the present invention can be used for delivering essentially any polyanionic molecule. As noted in the Background of the Invention, typical lipid-nucleic acid formulations are formed by combining the nucleic acid with a preformed cationic liposome (see, U.S. Pat. Nos. 4,897,355, 5,264,618, 5,279,833 and 5,283,185. In such methods, the nucleic acid is attracted to the cationic surface charge of the liposome and the resulting complexes are thought to be of the “sandwich-type” depicted in FIG. 1. As a result, a portion of the nucleic acid or plasmid remains exposed in serum and can be degraded by enzymes such as DNAse I. Others have attempted to incorporate the nucleic acid or plasmid into the interior of a liposome during formation. These methods typically result in the aggregation in solution of the cationic lipid-nucleic acid complexes (see FIG. 2). Passive loading of a plasmid into a preformed liposome has also not proven successful. Finally, the liposome-plasmid complexes which have been formed are typically 200 to 400 nm in size and are therefore cleared more rapidly from circulation than smaller sized complexes or particles. The present invention provides a method of preparing serum-stable plasmid-lipid particles in which the plasmid is encapsulated, in a lipid-bilayer and is protected from degradation. Additionally, the particles formed have a size of about 50 to about 150 nm, with a majority of the particles being about 65 to 85 nm. The particles can be formed by either a detergent dialysis method or by a modification of a reverse-phase method which utilizes organic solvents to provide a single phase during mixing of the components. Without intending to be bound by any particular mechanism of formation, FIG. 3 depicts a detergent dialysis approach to the formation of the plasmid-lipid particles. With reference to FIG. 3, a plasmid or other large nucleic acid is contacted with a detergent solution of cationic lipids to form a coated plasmid complex. These coated plasmids can aggregate and precipitate. However, the presence of a detergent reduces this aggregation and allows the coated plasmids to react with excess lipids (typically, non-cationic lipids) to form particles in which the plasmid is encapsulated in a lipid bilayer. As noted above, these particles differ from the more classical liposomes both in size (liposomes being typically 200-400 nm) in that there is little or no aqueous medium encapsulated by the particle's lipid bilayer. The methods described below for the formation of plasmid-lipid particles using organic solvents follow a similar scheme.
  • III. Methods of Forming Plasmid-Lipid Particles
  • The present invention provides methods for the formation of serum-stable plasmid-lipid particles. While the invention is described with reference to the use of plasmids, one of skill in the art will understand that the methods described herein are equally applicable to other larger nucleic acids or oligonucleotides. In one group of embodiments, the particles are formed using detergent dialysis. Thus, the present invention provides a method for the preparation of serum-stable plasmid-lipid particles, comprising:
      • (a) combining a plasmid with cationic lipids in a detergent solution to form a coated plasmid-lipid complex;
      • (b) contacting non-cationic lipids with the coated plasmid-lipid complex to form a detergent solution comprising a plasmid-lipid complex and non-cationic lipids; and
      • (c) dialyzing the detergent solution of step (b) to provide a solution of serum-stable plasmid-lipid particles, wherein the plasmid is encapsulated in a lipid bilayer and the particles are serum-stable and have a size of from about 50 to about 150 nm.
  • The plasmids which are useful in the present invention are typically nucleotide polymers which are to be administered to a subject for the purpose of repairing or enhancing the expression of a cellular protein. Accordingly, the nucleotide polymers can be polymers of nucleic acids including genomic DNA, cDNA, or mRNA. Still further, the plasmids may encode promoter regions, operator regions, structural regions. When nucleic acids other than plasmids are used the nucleic acids can contain nucleic acid analogs, for example, the antisense derivatives described in a review by Stein, et at., Science 261:1004-1011 (1993) and in U.S. Pat. Nos. 5,264,423 and 5,276,019, the disclosures of which are incorporated herein by reference.
  • The plasmids, or nucleic acids can be single-stranded DNA or RNA, or double-stranded DNA or DNA-RNA hybrid. Examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and self-replicating systems such as plasmid DNA.
  • Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to have prolonged activity, the single-stranded nucleic acids will preferably have some or all of the nucleotide linkages substituted with stable, non-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.
  • The nucleic acids used in the present invention will also include those nucleic acids in which modifications have been made in one or more sugar moieties and/or in one or more of the pyrimidine or purine bases. Examples of sugar modifications include replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, azido groups or functionalized as ethers or esters. Additionally, the entire sugar may be replaced with sterically and electronically similar structures, including aza-sugars and carbocyclic sugar analogs. Modifications in the purine or pyrimidine base moiety include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those of skill in the art.
  • Multiple genetic sequences can be also be used in the present methods. Thus, the sequences for different proteins may be located on one strand or plasmid. Promoter, enhancer, stress or chemically-regulated promoters, antibiotic-sensitive or nutrient-sensitive regions, as well as therapeutic protein encoding sequences, may be included as required. Non-encoding sequences may be also be present, to the extent they are necessary to achieve appropriate expression.
  • The nucleic acids used in the present method can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Generally, solid phase synthesis is preferred. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available. See, for example, Itakura, U.S. Pat. No. 4,401,796; Caruthers, et al., U.S. Pat. Nos. 4,458,066 and 4,500,707; Beaucage, et al., Tetrahedron Lett., 22:1859-1862 (1981); Matteucci; et al., J. Am. Chem. Soc., 103:3185-3191 (1981); Caruthers, et al., Genetic Engineering, 4:1-17 (1982); Jones, chapter 2, Atkinson, et al., chapter 3, and Sproat, et al., chapter 4, in Oligonucleotide Synthesis: A Practical Approach, Gait (ed.), IRL Press, Washington D.C. (1984); Froehler, et al., Tetrahedron Lett., 27:469-472 (1986); Froehler, et al., Nucleic Acids Res., 14:5399-5407 (1986); Sinha, et al. Tetrahedron Lett., 24:5843-5846 (1983); and Sinha, et al., Nucl. Acids Res., 12:4539-4557 (1984) which are incorporated herein by reference.
  • Cationic lipids which are useful in the present invention, include, for example, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. These lipids and related analogs, which are also useful in the present invention, have been described in co-pending U.S. Ser. No. 08/316,399; U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and 5,283,185, the disclosures of which are incorporated herein by reference. Additionally, a number of commercial preparations of cationic lipids are available and can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE® (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic liposomes comprising DOGS from Promega Corp., Madison, Wis., USA).
  • An initial solution of coated plasmid-lipid complexes is formed by combining the plasmid with the cationic lipids in a detergent solution. The detergent solution is preferably an aqueous solution of a neutral detergent having a critical micelle concentration of 15-300 mM, more preferably 20-50 mM. Examples of suitable detergents include, for example, N,N′-(octanoylimino)-bis-(trimethylene))-bis(D-gluconamide) (BIGCHAP); BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol) ether; Tween 20; Tween 40; Tween 60; Tween 80; Tween 85; Mega 8; Mega 9; Zwittergent® 3-08; Zwittergent® 3-10; Triton X-405; hexyl-, heptyl-, octyl- and nonyl-β-D-glucopyr and heptylthioglucopyranoside; with octyl β-D-glucopyranoside being the most preferred. The concentration of detergent in the detergent solution is typically about 100 mM to about 2 M, preferably from about 200 mM to about 1.5 M.
  • The cationic lipids and plasmid will typically be combined to produce a charge ratio (+/−) of about 1:1 to about 20:1, preferably in a ratio of about 1:1 to about 12:1, and more preferably in a ratio of about 2:1 to about 6:1. Additionally, the overall concentration of plasmid in solution will typically be from about 25 μg/mL to about 1 mg/mL, preferably from about 25 μg/mL to about 200 μg/mL, and more preferably from about 50 μg/mL to about 100 μg/mL. The combination of plasmids and cationic lipids in detergent solution is kept, typically at room temperature, for a period of time which is sufficient for the coated complexes to form. Alternatively, the plasmids and cationic lipids can be combined in the detergent solution and warmed to temperatures of up to about 37° C. For plasmids which are particularly sensitive to temperature, the coated complexes can be formed at lower temperatures, typically down to about 4° C.
  • The detergent solution of the coated plasmid-lipid complexes is then contacted with non-cationic lipids to provide a detergent solution of plasmid-lipid complexes and non-cationic lipids. The noncationic lipids which are useful in this step include, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cardiolipin, and cerebrosides. In preferred embodiments, the non-cationic lipids are diacylphosphatidylcholine, diacylphosphatidylethanolamine, cerarnide or sphingomyelin. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains. More preferably the acyl groups are lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl. In particularly preferred embodiments, the nonmtionic lipid will be 1,2-sn-dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC) or egg phosphatidylcholine (EPC). In the most preferred embodiments, the plasmid-lipid particles will be fusogenic particles with enhanced properties in vivo and the non-cationic lipid will be DOPE. In other preferred embodiments, the non-cationic lipids will further comprise polyethylene glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycol conjugated to ceramides, as described in co-pending U.S. Ser. No. 08/316,429, incorporated herein by reference.
  • The amount of non-cationic lipid which is used in the present methods is typically about 2 to about 20 mg of total lipids to 50 μg of plasmid. Preferably the amount of total lipid is from about 5 to about 10 mg per 50 μg of plasmid.
  • Following formation of the detergent solution of plasmid-lipid complexes and non-cationic lipids, the detergent is removed, preferably by dialysis. The removal of the detergent results in the formation of a lipid-bilayer which surrounds the plasmid providing serum-stable plasmid-lipid particles which have a size of from about 50 nm to about 150 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.
  • The serum-stable plasmid-lipid particles can be sized by any of the methods available for sizing liposomes. The sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes.
  • Several techniques are available for sizing the particles to a desired size. One sizing method, used for liposomes and equally applicable to the present particles is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a particle suspension either by bath or probe sonication produces a progressive size reduction down to particles of less than about 50 nm in size. Homogenization is another method which relies on shearing energy to fragment larger particles into smaller ones. In a typical homogenization procedure, particles are recirculated through a standard emulsion homogenizer until selected particle sizes, typically between about 60 and 80 nm, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination, or QELS.
  • Extrusion of the particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. The particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.
  • In another group of embodiments, the present invention provides a method for the preparation of serum-stable plasmid-lipid particles, comprising;
      • (a) preparing a mixture comprising cationic lipids and non-cationic lipids in an organic solvent;
      • (b) contacting an aqueous solution of nucleic acid with said mixture in step (a) to provide a clear single phase; and
      • (c) removing said organic solvent to provide a suspension of plasmid-lipid particles, wherein said plasmid is encapsulated in a lipid bilayer, and said particles are stable in serum and have a size of from about 50 to about 150 nm.
  • The plasmids (or nucleic acids), cationic lipids and non-cationic lipids which are useful in this group of embodiments are as described for the detergent dialysis methods above.
  • The selection of an organic solvent will typically involve consideration of solvent polarity and the ease with which the solvent can be removed at the later stages of particle formation. The organic solvent, which is also used as a solubilizing agent, is in an amount sufficient to provide a clear single phase mixture of plasmid and lipids. Suitable solvents include chloroform, dichloromethane, diethylether, cyclohexane, cyclopentane, benzene, toluene, methanol, or other aliphatic alcohols such as propanol, isopropanol, butanol, tert-butanol, iso-butanol, pentanol and hexanol. Combinations of two or more solvents may also be used in the present invention.
  • Contacting the plasmid with the organic solution of cationic and non-cationic lipids is accomplished by mixing together a first solution of plasmid, which is typically an aqueous solution and a second organic solution of the lipids. One of skill in the art will understand that this mixing can take place by any number of methods, for example by mechanical means such as by using vortex mixers.
  • After the plasmid has been contacted with the organic solution of lipids, the organic solvent is removed, thus forming an aqueous suspension of serum-stable plasmid-lipid-particles. The methods used to remove the organic solvent will typically involve evaporation at reduced pressures or blowing a stream of inert gas (e.g., nitrogen or argon) across the mixture.
  • The serum-stable plasmid-lipid particles thus formed will typically be sized from about 50 nm to 150 nm. To achieve further size reduction or homogeneity of size in the particles, sizing can be conducted as described above.
  • In other embodiments, the methods will further comprise adding nonlipid polycations which are useful to effect the transformation of cells using the present compositions. Examples of suitable nonlipid polycations include, hexadimethrine bromide (sold under the brand name POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of heaxadimethrine. Other suitable polycations include, for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine and polyethyleneimine.
  • In other embodiments, the polyoxyethylene conjugates which are used in the plasmid-lipid particles of the present invention can be prepared by combining the conjugating group (i.e. phosphatidic acid or phosphatidylethanolamine) with an appropriately functionalized polyoxyethylene derivative. For example, phosphatddylethanolamine can be combined with polyoxyethylene bis(p-toluenesulfonate) to provide a phosphatidylethanolamine-polyoxyethylene conjugate. See, Woodle, et al., Biochim. Biophys. Acta 1105:193-200 (1992), incorporated herein by reference.
  • The present invention also provides plasmid-lipid particles which are prepared by the methods described above. In preferred embodiments, the particles comprise a plasmid, a non-cationic lipid which is a mixture of POPC and PEG-Cer or DOPE and PEG-Cer, and a cationic lipid which is DODAC.
  • IV. Pharmaceutical Preparations
  • The plasmid-lipid particles of the present invention can be administered either alone or in mixture with a physiologically-acceptable carrier (such as physiological saline or phosphate buffer) -selected in accordance with the route of administration and standard pharmaceutical practice.
  • Pharmaceutical compositions comprising the plasmid-lipid particles of the invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier. Generally, normal saline will be employed-as the pharmaceutically acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. These compositions may be sterilized by conventional, well known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Additionally, the particle suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • The concentration of particles in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, particles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration. For diagnosis, the amount of particles administered will depend upon the particular label used, the disease state being diagnosed and the judgement of the clinician but will generally be between about 0.01 and about 50 mg per kilogram of body weight, preferably between about 0.1 and about 5 mg/kg of body weight.
  • As noted above, it is often desirable to include polyethylene glycol (PEG), PEG-ceramide, or ganglioside GM1-modified lipids to the particles. Addition of such components prevents particle aggregation and provides a means for increasing circulation lifetime and increasing the delivery of the plasmid-lipid particles to the target tissues. Typically, the concentration of the PEG, PEG-ceramide or GM1-modified lipids in the particle will be about 1-15%.
  • Overall particle charge is also an important determinant in particle clearance from the blood, with negatively charged complexes being taken up more rapidly by the reticuloendothelial system (Juliano, Biochem. Biophys. Res. Commun. 63:651 (1975)) and thus having shorter half-lives in the bloodstream. Particles with prolonged circulation half-lives are typically desirable for therapeutic and diagnostic uses. For instance, particles which can be maintained from 8, 12, or up to 24 hours in the bloodstream are particularly preferred.
  • In another example of their use, plasmid-lipid particles can be incorporated into a broad range of topical dosage forms including but not limited to gels, oils, emulsions and the like. For instance, the suspension containing the plasmid-lipid particles can be formulated and administered as topical creams, pastes, ointments, gels, lotions and the like.
  • The present invention also provides plasmid-lipid particles in kit form. The kit will typically be comprised of a container which is compartmentalized for holding the various elements of the kit. The kit will contain the compositions of the present inventions, preferably in dehydrated form, with instructions for their rehydration and administration. In still other embodiments, the particles and/or compositions comprising the particles will have a targeting moiety attached to the surface of the particle. Methods of attaching targeting moieties (e.g., antibodies, proteins) to lipids (such as those used in the present particles) are known to those of skill in the art.
  • Dosage for the plasmid-lipid particle formulation will depend on the ratio of nucleic acid to lipid and the administrating physician's opinion based on age, weight, and condition of the patient.
  • V. Administration of Plasmid-Lipid Particle Formulations
  • The serum-stable plasmid-lipid particles of the present invention are useful for the introduction of plasmids into cells. Accordingly, the present invention also provides methods for introducing a plasmid into a cell. The methods are carried out in vitro or in vivo by first forming the particles as described above, then contacting the particles with the cells for a period of time sufficient for transfection to occur.
  • The particles of the present invention can be adsorbed to almost any cell type. Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells. Transfer or incorporation of the nucleic acid portion of the particle can take place via any one of these pathways. In particular, when fusion takes place, the particle membrane is integrated into the cell membrane and the contents of the particle combine with the intracellular fluid. Contact between the cells and the plasmid-lipid particles, when carried out in vitro, will take place in a biologically compatible medium. The concentration of particles can vary widely depending on the particular application, but is generally between about 1 μmol and about 10 mmol. Treatment of the cells with the plasmid-lipid particles will generally be carried out at physiological temperatures (about 37° C.) for periods of time of from about 1 to 6 hours, preferably of from about 2 to 4 hours. For in vitro applications, the delivery of nucleic acids can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type. In preferred embodiments, the cells will be animal cells, more preferably mammalian cells, and most preferably human cells.
  • In one group of preferred embodiments, a plasmid-lipid particle suspension is added to 60-80% confluent plated cells having a cell density of from about 103 to about 105 cells/mL, more preferably about 2×104 cells/mL. The concentration of the suspension added to the cells is preferably of from about 0.01 to 0.2 μg/mL, more preferably about 0.1 μg/mL.
  • Typical applications include using well known transfection procedures to provide intracellular delivery of DNA or mRNA sequences which code for therapeutically useful polypeptides. However, the compositions can also be used for the delivery of the expressed gene product or protein itself. In this manner, therapy is provided for genetic diseases by supplying deficient or absent gene products (i.e., for Duchenne's dystrophy, see Kunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989), and for cystic fibrosis, see Goodfellow, Nature 341:102-103 (1989)). Other uses for the compositions of the present invention include introduction of antisense oligonucleotides in cells (see, Bennett, et al., Mol. Phamn. 41:1023-1033 (1992)).
  • Alternatively, the compositions of the present invention can also be used for the transfection of cells in vivo, using methods which are known to those of skill in the art. In particular, Zhu, et al., Science 261:209-211 (1993), incorporated herein by reference, describes the intravenous delivery of cytomegalovirus (CMV)-chloramphenicol acetyltransferase (CAT) expression plasmid using DOTMA-DOPE complexes. Hyde, et al., Nature 362:250-256 (1993), incorporated herein by reference, describes the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to epithelia of the airway and to alveoli in the lung of mice, using liposomes. Brigham, et al., Am. J. Med. Sci. 298:278-281 (1989), incorporated herein by reference, describes the in vivo transfection of lungs of mice with a functioning prokaryotic gene encoding the intracellular enzyme, chloramphenicol acetyltransferase (CAT).
  • For in vivo administration, the pharmaceutical compositions are preferably administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. For example, see Stadler, et al., U.S. Pat. No. 5,286,634, which is incorporated herein by reference. Intracellular nucleic acid delivery has also been discussed in Straubringer, et al., METHODS IN ENZYMOLOGY, Academic Press, New York. 101:512-527 (1983); Mannino, et al., Biotechniques 6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. Drug Carrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278 (1993). Still other methods of administering lipid-based therapeutics are described in, for example, Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
  • In other methods, the pharmaceutical preparations may be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical, “open” or “closed” procedures. By “topical”, it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. “Open” procedures are those procedures which include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue. “Closed” procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical preparations may be administered to the meninges or spinal cord by infusion during a lumbar puncture followed by appropriate positioning of the patient as commonly practiced for spinal anesthesia or metzamide imaging of the spinal cord. Alternatively, the preparations may be administered through endoscopic devices.
  • The plasmid-lipid particles can also be administered in an aerosol inhaled into the lungs (see, Brigham, et al., Am. J. Sci. 298(4):278-281 (1989)) or by direct injection at the site of disease (Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)).
  • The methods of the present invention may be practiced in a variety of hosts. Preferred hosts include mammalian species, such as humans, non-human primates, dogs, cats, cattle, horses, sheep, and the like.
  • VI. EXAMPLES
  • The following examples are offered solely for the purposes of illustration, and are intended neither to limit nor to define the invention. In each of these examples, the term “DNA” or “plasmid” refers to the plasmid pCMV4-CAT.
  • Example 1
  • This example illustrates the encapsulation of a plasmid in a lipid bilayer system using either a reverse-phase method or a detergent dialysis method.
  • Reverse Phase Method
  • pCMV4-CA-T plasmid (50 μg) was encapsulated in a lipid bilayer which was constructed using 20 mg POPC:PEG-Cer-C20 (95:5 mole % ratio) with between 0 and 0.3 mg DODAC. The encapsulation method utilized a modification of the classical reverse phase method for entrapment. Specifically, 1.050 mL of chloroform:methanol in a 1:2.1 mole % ratio was added to a lipid film containing 2 μL of 14C-cholesteryl hexadecyl ether (6.66 μL/μCi). This was followed by the addition of 220 μL H2O and 33 μL 3H-pCMV4-CAT plasmid (158,000 dpm/μL; 1.5 mg/mL). This combination provided a clear single phase. The CHCl3 and most of the methanol were removed under a stream of nitrogen while vortexing the mixture. The resulting 250 μL suspension of encapsulated plasmid was diluted with 1 mL of H2O and extruded 5 times through one 400 nm filter followed by extrusion 5 times through one 200 nm filter. The resulting vesicle size was approximately 150 to 200 nm in diameter.
  • Detergent Dialysis Method
  • pCMVCAT (50 μg consisting of 20 μL of 3H-pCMV4-CAT and 30 μL of cold pCMV4-CAT at a concentration of 1 mg/mL, “plasmid”) was incubated with DODAC at various DODAC:plasmid charge ratios in 100 μL of 1M n-octyl-β-D-glucopyranoside and 400 μL H2O for 30 min at room temperature. The resulting plasmid:DODAC mixture was added to a suspension of 5 mg POPC:PEG-Cer(C20)or 10 mg DOPE:PEG-Cer(C20) (containing 1 μL 14C-cholesteryl hexadecyl ether; 6.66 μL/μCi) in 100 μL of 1 M n-octyl-β-D-glucopyranoside and 400 μL of H2O). The amounts used for each lipid to achieve a desired charge ratio are shown in Tables 1-3. The suspension was dialysed against HBS at pH 7.4 overnight. The resulting encapsulated plasmid can be used without further sizing.
    TABLE 1
    Calculation of % DODAC in vesicles for any given DODAC:DNA charge ratio
    as a function of total mg of lipid
    DNA POPC DODAC PEGCer POPC DODAC PEGCer total lipid
    Lipid (mg) DODAC:DNA microgram % % % mg mg mg micromole
    20 1 50 89.25979 0.740207 10 14.09342 0.0895 5.817079 20.77528
    20 2 50 88.52161 1.478393 10 13.99597 0.179 5.82503 20.80368
    20 3 50 87.78543 2.214567 10 13.89852 0.2685 5.83298 20.83207
    20 4 50 87.05126 2.948737 10 13.80107 0.358 5.840931 20.86047
    20 5 50 86.31909 3.68091 10 13.70362 0.4475 5.848882 20.88886
    20 6 50 85.5889 4.411096 10 13.60617 0.537 5.856832 20.91726
    20 7 50 84.8607 5.139302 10 13.50872 0.6265 5.864783 20.94565
    20 8 50 84.13446 5.865537 10 13.41127 0.716 5.872734 20.97405
    20 9 50 83.41019 6.589808 10 13.31382 0.8055 5.880684 21.00244
    20 10 50 82.68788 7.312122 10 13.21637 0.895 5.888635 21.03084
    20 11 50 81.96751 8.03249 10 13.11891 0.9845 5.896585 21.05923
    20 12 50 81.24908 8.750917 10 13.02146 1.074 5.904536 21.08763
    20 13 50 80.53259 9.467412 10 12.92401 1.1635 5.912487 21.11602
    20 14 50 79.81802 10.18198 10 12.82656 1.253 5.920437 21.14442
    20 15 50 79.10536 10.89464 10 12.72911 1.3425 5.928388 21.17281
    20 16 50 78.39462 11.60538 10 12.63166 1.432 5.936339 21.20121
    20 17 50 77.68578 12.31422 10 12.53421 1.5215 5.944289 21.2296
    20 18 50 76.97883 13.02117 10 12.43676 1.611 5.95224 21.258
    20 19 50 76.27376 13.72624 10 12.33931 1.7005 5.96019 21.28639
    20 20 50 75.57058 14.42942 10 12.24186 1.79 5.968141 21.31479
  • TABLE 2
    Calculation of % DODAC in vesicles for any given DODAC:DNA charge ratio
    as a function of total mg of lipid
    DNA POPC DODAC PEGCer POPC DODAC PEGCer total lipid
    Lipid (mg) DODAC:DNA microgram % % % mg mg mg micromole
    5 1 50 87.05126 2.948737 10 3.450267 0.0895 1.460233 5.215117
    5 2 50 84.13446 5.865537 10 3.352817 0.179 1.468183 5.243512
    5 3 50 81.24908 8.750917 10 3.255366 0.2685 1.476134 5.271907
    5 4 50 78.39462 11.60538 10 3.157915 0.358 1.484085 5.300302
    5 5 50 75.57058 14.42942 10 3.060465 0.4475 1.492035 5.328697
    5 6 50 72.77647 17.22353 10 2.963014 0.537 1.499986 5.357092
    5 7 50 70.01183 19.98817 10 2.865563 0.6265 1.507937 5.385488
    5 8 50 67.27619 22.72381 10 2.768113 0.716 1.515887 5.413883
    5 9 50 64.56909 25.43091 10 2.670662 0.8055 1.523838 5.442278
    5 10 50 61.8901 28.1099 10 2.573212 0.895 1.531788 5.470673
    5 11 50 59.23877 30.76123 10 2.475761 0.9845 1.539739 5.499068
    5 12 50 56.61469 33.38531 10 2.37831 1.074 1.54769 5.527463
    5 13 50 54.01742 35.98258 10 2.28086 1.1635 1.55564 5.555858
    5 14 50 51.44657 38.55343 10 2.183409 1.253 1.563591 5.584253
    5 15 50 48.90173 41.09827 10 2.085959 1.3425 1.571541 5.612648
    5 16 50 46.38252 43.61748 10 1.988508 1.432 1.579492 5.641043
    5 17 50 43.88853 46.11147 10 1.891057 1.5215 1.587443 5.669438
    5 18 50 41.41941 48.58059 10 1.793607 1.611 1.595393 5.697833
    5 19 50 38.97477 51.02523 10 1.696156 1.7005 1.603344 5.726228
    5 20 50 36.55426 53.44574 10 1.598705 1.79 1.611295 5.754623
  • TABLE 3
    Calculation of % DODAC in vesicles for any given DODAC:DNA charge ratio
    as a function of total mg of lipid
    DNA DOPE DODAC PEGCer DOPE DODAC PEGCer total lipid
    Lipid (mg) DODAC:DNA microgram % % % mg mg mg micromole
    10 1 50 88.54333 1.456667 10 6.954544 0.0895 2.955956 10.55698
    10 2 50 87.09389 2.906111 10 6.857699 0.179 2.963301 10.58322
    10 3 50 85.65161 4.348388 10 6.760853 0.2685 2.970647 10.60945
    10 4 50 84.21645 5.783549 10 6.664007 0.358 2.977993 10.63569
    10 5 50 82.78835 7.211648 10 6.567162 0.4475 2.985338 10.66192
    10 6 50 81.36726 8.632736 10 6.470316 0.537 2.992684 10.68816
    10 7 50 79.95314 10.04686 10 6.37347 0.6265 3.00003 10.71439
    10 8 50 78.54591 11.45409 10 6.276625 0.716 3.007375 10.74063
    10 9 50 77.14555 12.85445 10 6.179779 0.8055 3.014721 10.76686
    10 10 50 75.752 14.248 10 6.082933 0.895 3.022067 10.7931
    10 11 50 74.3652 15.6348 10 5.986088 0.9845 3.029412 10.81933
    10 12 50 72.98511 17.01489 10 5.889242 1.074 3.036758 10.84556
    10 13 50 71.61168 18.38832 10 5.792396 1.1635 3.044104 10.8718
    10 14 50 70.24487 19.75513 10 5.69555 1.253 3.05145 10.89803
    10 15 50 68.88462 21.11538 10 5.598705 1.3425 3.058795 10.92427
    10 16 50 67.53089 22.46911 10 5.501859 1.432 3.066141 10.9505
    10 17 50 66.18362 23.81638 10 5.405013 1.5215 3.073487 10.97674
    10 18 50 64.84279 25.15721 10 5.308168 1.611 3.080832 11.00297
    10 19 50 63.50833 26.49167 10 5.211322 1.7005 3.088178 11.02921
    10 20 50 62.1802 27.8198 10 5.114476 1.79 3.095524 11.05544
  • Example 2
  • This example illustrates the level of plasmid “protection” from external medium using anion exchange chromatography.
  • The extent of encapsulation or protection of the plasmid from the external medium was assessed by anion exchange chromatography as follows: a 50 μL aliquot of each sample was eluted on a DEAE Sepharose CL-6B column and the fractions were assessed for both 3H-plasmid and 14C-lipid by scintillation counting. Any exposed negative charges, such as those present on DNA molecules will bind to the anion exchange column and will not elute with the 14C-lipid. DNA which has its negative charge “protected” or nonexposed will not bind to the ion exchange resin and will elute with the 14C-lipid.
  • Reverse Phase Method particles with POPC:DODAC:PEG-Cer(C20)
  • FIG. 4 presents the results describing the relationship between DODAC:plasmid charge ratio (see Table 1 for amounts of POPC, DODAC and PEG-Cer(C20, using 20 mg total lipid) and percent recovery of plasmid after extrusion through a 400 nm filter and a 200 nm filter. An increase in percent plasmid recovered was observed corresponding to an increase in DODAC:plasmid charge ratio. No plasmid was recovered in the absence of DODAC while, at a DODAC:plasmid charge ratio of 2:1, 90% of the plasmid was recovered after extrusion through a 400 nm filter and 70% of the plasmid was recovered after extrusion through a 200 nm filter. Nearly 100% of the plasmid recovered from extrusion through a 200 nm filter was recovered by anion exchange chromatography (see FIG. 5) suggesting that all of the recovered plasmid was encapsulated. This corresponded to an overall encapsulation efficiency of about 70%. Lipid recoveries after extrusion and anion exchange chromatography were 90% after extrusion through a 400 nm filter and 70% after extrusion through a 200 nm filter (see FIG. 6). Of the 70% lipid recovered after extrusion through a 200 nm filter, nearly 100% was recovered after anion exchange chromatography (see FIG. 7). Lipid and plasmid recovery after extrusion and anion exchange chromatography were nearly identical.
  • Dialysis Method (Particles with POPC:DODAC:PEG-Cer(C20)
  • FIG. 8 provides the results which illustrate the effect of DODAC:plasmid charge ratio on the percent recovery of lipid and plasmid from anion exchange chromatography following preparation of the particles using the detergent dialysis method of Example 1 (amounts of lipids are provided in Table 2 for 5 mg total lipid compositions). Significant protection was observed over a DODAC:plasmid charge ratio of about 3:1 to 5:1. Also, it appears that significant protection of the plasmid is achieved at a DODAC:plasmid charge ratio of about 8:1. The recovery of lipid decreased from 100% in the absence of DODAC to about 85% at a DODAC:plasmid charge ratio of 8:1.
  • Dialysis Method (Particles with DOPE:DODAC:PEG-Cer(C20)
  • In a similar manner to that described for the POPC-containing particles, the fusogenic lipid composition DOPE:DODAC:PEG-Cer(C20) was assessed by anion exchange chromatography. Aliquots (50 μL) of the plasmid-lipid particles (prepared by detergent dialysis, using the amounts provided in Table 3) were eluted on a DEAE Sepharose CL-6B column. FIG. 9 provides the results and illustrates the relationship between the DODAC:DNA charge ratio and % recovery of lipid and DNA for particles using 10 mg of total lipid. DNA encapsulation occurred at a DODAC:DNA charge ratio of 4:1.
  • Example 3
  • This example illustrates the serum stability achieved using plasmid:lipid particles prepared by the methods of Example 1.
  • To establish the serum stability of the plasmid:lipid particles, aliquots of the particle mixtures prepared according to both the reverse phase and dialysis methods of Example 1 were incubated in mouse serum (Cedar Lane) for 15 min and for 30 min at 37° C. Prior to incubation, the lipid associated plasmid was eluted on a DEAE Sepharose CL-6B column to remove unencapsulated plasmid. Following incubation, an aliquot of the incubation mixture was eluted in HBS on a Sepharose CL-4B column.
  • As a control, 1.5 mg of free 3H-pCMV4-CAT was eluted on a Sepharose CL-4B column in HBS, pH 7.4 (see FIG. 10). For comparison, 1.5 mg of free 3H-pCMV4-CAT was incubated in 500 μL of mouse serum at 37° C. for 30 min and eluted in the same manner (see FIG. 11). Note that in FIG. 10, the free plasmid eluted in the void volume of the column while, in FIG. 11, the plasmid incubated in serum eluted in the included volume suggesting that the plasmid had been digested by serum enzymes.
  • Serum Stability of Plasmid-Lipid Particles Prepared by Reverse Phase
  • (particles prepared from POPC:DODAC:PEG-Cer(C20))
  • The stability of plasmid-lipid particles was assessed by incubation of a 50 μL aliquot in 500 μL of mouse serum (Cedar Lane) for 15 min at 37° C. A 500 μL aliquot of the incubation mixture was eluted in HBS on a Sepharose CL-4B column (see FIG. 12). Comigration of the plasmid and lipid in the void volume strongly suggests that no plasmid degradation has occurred. Any serum associated plasmid or lipid should have been detected as a peak at around fraction 35 (see control results in FIG. 11).
  • Serum Stability of Plasmid-Lipid Particles Prepared by Dialysis
  • (particles prepared from DOPE:DODAC:PEG-Cer(C20))
  • A 50 μL aliquot of a particle suspension prepared at a DODAC:plasmid charge ratio of 4:1 was incubated in 500 μL of mouse serum at 37° C. for 30 min and eluted on a Sepharose CL-4B column as described above. FIG. 13 shows the elution profile of the sample after incubation in serum. As can be seen in FIG. 13, 94% of the plasmid is recovered in the void volume suggesting that essentially all of the plasmid recovered from anion exchange chromatography is encapsulated.
  • Example 4
  • This example illustrates the level of plasmid encapsulated in lipid bilayers. Empty lipid complexes containing an aqueous space are relatively low in density and have a tendency to equilibrate nearer the top of a density gradient. Free plasmid is relatively high in density and will therefore equilibrate at a position nearer the bottom of the density gradient (where the Ficoll concentration is highest; more dense). Encapsulated plasmid will equilibrate on the gradient at a position between the positions of the empty lipid complexes and free plasmid.
  • Reverse Phase Methods (POPC:DODAC:PEG-Cer(C20)
  • A 100 μL aliquot of lipid complexes prepared as above but in the absence of plasmid was added to a Ficoll 400 continuous density gradient (0-7.5%) prepared in HBS (pH 7.4). Similarly a 50 μL aliquot of the plasmid:lipid particle suspension and 0.25 μL of free 3H-pCMV4-CAT was added to two separate Ficoll 400 gradients. The samples were centrifuged at 100,000 ×g for 21 hours. 3H-pCMV4-CAT and 14C-lipid was assessed in 250 μL aliquots in each gradient by scintillation counting.
  • The empty lipid complexes exhibited a broad range of densities peaking at approximately fraction 25 (see FIG. 14). The broad range was probably due to heterogeneity in lipid complex or liposome size and lamellarity since the complexes were only extruded three times through one 200 nm filter, rather than the usual ten times through two 100 nm filters. The free plasmid was present as a single peak near the bottom of the gradient near fraction 35 (see FIG. 15). The gradient profile of the plasmid:lipid particle suspension suggested an association of plasmid with the lipid as there was comigration of the plasmid and the lipid and the densities of both were markedly different from that of their free counterparts (see FIG. 16). The plasmid:lipid ratio was not constant over the gradient profile which can be explained by assuming that not all plasmid-lipid particles contained the same number of plasmid molecules.
  • Dialysis Methods (DOPE:DODAC:PEG-Cer(C20)
  • Plasmid-lipid particles were prepared as described in Example 1 using detergent dialysis with a lipid composition of DOPE:DODAC:PEG-Cer(C20) (83.5:6.5:10 mole %). The particles were subjected to density gradient centrifugation as described for the plasmid-lipid particles prepared by reverse phase methods. The empty lipid complexes were present as a single peak at about fraction 20 (see FIG. 17). Free plasmid was present at about fraction 31. It was evident from these controls that successful entrapment of the pCMV4-CAT was achieved as determined by the comigration of lipid and plasmid with a peak between that of the free lipid and plasmid controls.
  • Example 5
  • This example illustrates the size distribution of plasmid-lipid particles as measured by quasielastic light scattering using a Nicomp Submicron Particle Sizer.
  • Plasmid-lipid particles were prepared by detergent dialysis as described in Example 1. The lipid composition was DOPE:DODAC:PEG-Cer(C20) (83.5:6.5:10 mole The particles were sized using a Nicomp Submicron Particle Sizer (see FIG. 18).
  • The log normal distribution exhibited a x2 of 0.2, indicating an extremely homogeneous distribution. The mean diameter of the particles with entrapped pCMV4-CAT plasmid was 72.4 nm.
  • Example 6
  • This example illustrates the clearance and in vivo transfection of plasmid:lipid particles in mice.
  • Reverse Phase (POPC:DODAC:PEG-Cer(C20))
  • Encapsulated plasmid blood clearance was tested in three IRC mice as a function of percent recovered dose over time. Percent recovery of free 3H-plasmid was plotted over a similar time course as a control (see FIG. 19). The encapsulated plasmid exhibits a clearance rate which is much slower than that of the free 3H-plasmid. Additionally, the plasmid:lipid ratio does not change significantly over the time course of the experiment confirming that the plasmid clearance rate is associated with the clearance rate of the lipid carrier itself.
  • Detergent Dialysis (DOPE:DODAC:PEG-Cer(C14 or C20)
  • Fusogenic particles of pCMV4-CAT encapsulated in DOPE:DODAC:PEG-Cer(C14 or C20)(83.5:6.5:10 mole %) were prepared as follows:
  • pCMV4-CAT (50 μg)(42 μL of 3H-pCMV4-CAT; 108 dpm/μL, 1.19 mg/mL) was incubated with DODAC (407 μg; ˜4:1 DODAC:DNA charge ratio) in 100 μL of 1 M OGP and 400 μL of water for 30 min at room temperature. This DNA:DODAC complex mixture was added to a suspension of 10 mg of DOPE:PEG-Cer(C14 or C20) (63.5:10 mole %) and the particles were constructed as described in Example 1 (detergent dialysis). The plasmid-lipid particles for blood clearance studies contained 0.75 μL of 14C-cholesteryl hexadecyl ether (CHE) (6.66 μL/μCi) in 100 μL of 1 M OGP and 400 μL of water. Control particles prepared without DNA contained 2 μL of 14C-CHE for the particles containing PEG-Cer(C14) and 0.75 μL of 14C-CHE for the particles containing PEG-Cer(C20). For in vivo transfection, no 14C-lipid label was used as it would interfere with the CAT assay.
  • Clearance of pCMV4-CAT “DNA” encapsulatedin DOPE:DODAC:PEG-Cer (C14 and C20)
  • External “untrapped” DNA was removed by anion exchange chromatography using DEAE Sepharose CL-6B prior to injection into mice. Encapsulation efficiencies were approximately 42% for the systems containing PEG-Cer(C20) and 60% for the systems containing PEG-Cer(C14).
  • Three groups of three female ICR mice (20-25 g) were injected with 200 μL of DNA-encapsulated DOPE:DODAC:PEG-Cer(C20) ((83.5:6.5:10 mole %) each and another set of nine mice were injected with 200 μL of DNA-encapsulated DOPE:DODAC:PEG-Cer(C14) ((83.5:6.5:10 mole %) each. One group of mice was sacrificed and blood was taken at each of three time points (1, 2 and 5 hours). The plasma was separated from whole blood by centrifugation in 0.5 mL EDTA coated Tainer tubes. A 200 μL aliquot of the plasma from each mouse was assayed for 3H-DNA and 14C-lipid by scintillation counting.
  • Control particles (no DNA) which had been passed down an anion exchange column also were also analyzed. Two hundred microliters each of DOPE:DODAC:PEG-Cer(C20) control particles was injected into one group of three female ICR mice and 200 μL of DOPE:DODAC:PEG-Cer(C14) control particles was injected into three groups of three of the ICR mice. The plasma was analyzed for 14C-lipid after 1, 2 and 5 hours.
  • FIG. 20 shows the clearance of DNA encapsulated in particles composed of DOPE:DODAC:PEG-Cer(C20) ((83.5:6.5:10 mole %). The DNA and lipid are cleared much less rapidly from the circulation than when PEG-Cer(C14) is used (see FIG. 21). Nearly 50% of the lipid and DNA are present after 1 hour. A significant amount of DNA and lipid were still present after 5 hr. The amount of DNA and lipid injected was 1.8 μg and 853 μg, respectively. Control particles exhibited a clearance similar to that of the plasmid-lipid particles.
  • FIG. 21 shows the clearance of DNA encapsulated in particles composed of DOPE:DODAC:PEG-Cer(C14) ((83.5:6.5:10 mole %). Both DNA and lipid are cleared rapidly from the circulation with only about 20% of the lipid and 10% of the DNA present in the plasma after 1 hr. The amount of DNA and lipid injected was 2.7 μg and 912 μg, respectively. Control particles exhibited a clearance similar to that of the plasmid-lipid particles.
  • In Vivo Transfection in Lung, Liver and Spleen
  • Three groups of four IRC mice were injected via tail vein with pCMV4-CAT encapsulated in lipid particles composed of DOPE:DODAC:PEG-Cer(C14) (83.5:6.5:10 mole %, “A”) or DOPE:DODAC:PEG-Cer(C20) (83.5:6.5:10 mole %, “B”), prepared as described above. The mice were sacrificed after 2, 4 and 8 days and the lung, liver and spleen were assayed for CAT activity according to a modification of Deigh, Anal. Biochem. 156:251-256 (1986). The amount of plasmid injected was 2.6 μg for the particles containing PEG-Cer(C14) and 1.5 μg for the particles containing PEG-Cer(C20).
  • FIG. 22 shows the results of in vivo transfection achieved in the lung. As can be seen from this figure, treatment with formulation “A” provided excellent transfection efficiency (based on CAT activity) up to 4 days. Formulation “B”, while resulting in overall lower levels of CAT activity, provided relatively constant levels of enzyme activity over 8 days.
  • FIG. 23 shows the results of transfection achieved in the liver. For both formulations, transfection (and CAT activity) reached a maximum at 4 days.
  • FIG. 24 shows the results of transfection achieved in the spleen wherein the maximum transfection was found for both formulations to occur after 2 days.
  • VII. Conclusion
  • As discussed above, in accordance with one of its aspects, the present invention provides methods for preparing serum-stable plasmid-lipid particles which are useful for the transfection of cells; both in vitro and in vivo.
  • All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
  • Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (23)

1. A method for the preparation of serum-stable plasmid-lipid particles, comprising:
(a) combining a plasmid with cationic lipids in a detergent solution to provide a coated plasmid-lipid complex;
(b) contacting non-cationic lipids with said coated plasmid-lipid complex to provide a solution comprising detergent, a plasmid-lipid complex and non-cationic lipids; and
(c) removing said detergent from said solution of step (b) to provide a solution of serum-stable plasmid-lipid particles, wherein said plasmid is encapsulated in a lipid bilayer and said particles are serum-stable and have a size of from about 50 to about 150 nm.
2. A method in accordance with claim 1, wherein said removing is by dialysis.
3. A method in accordance with claim 1, wherein step (b) further comprises adding a polyethylene glycol-lipid conjugate.
4. A method in accordance with claim 2, 3, wherein said polyethylene glycol-lipid conjugate is a PEG-ceramide conjugate.
5. A method in accordance with claim 1, further comprising;
(d) sizing said particles to achieve a uniform particle size.
6. A method in accordance with claim 1, wherein said cationic lipids are selected from the group consisting of DODAC, DDAB, DOTAP, DOTMA, DOSPA, DOGS, DC-Chol and combinations thereof.
7. A method in accordance with claim 1, wherein said non-cationic lipids are selected from the group consisting of DOPE, POPC, EPC and combinations thereof.
8. A method in accordance with claim 1, wherein said detergent solution comprises a detergent having a critical micelle concentration of between about 20 mM and 50 mM.
9. A method in accordance with claim 8, wherein said detergent is n-octyl-β-D-glucopyranoside.
10. A method for the preparation of serum-stable plasmid-lipid particles, comprising;
(a) preparing a mixture comprising cationic lipids and non-cationic lipids in an organic solvent;
(b) contacting an aqueous solution of plasmid with said mixture prepared in step (a) to provide a clear single phase; and
(c) removing said organic solvent to provide a suspension of plasmid-lipid particles, wherein said plasmid is encapsulated in a lipid bilayer, and said particles are stable in serum and have a size of from about 50 to about 150 nm.
11. A method in accordance with claim 10, wherein said non-cationic lipids comprise a polyethylene glycol-lipid conjugate.
12. A method in accordance with claim 11, wherein said polyethylene glycol-lipid conjugate is a PEG-ceramide conjugate.
13. A method in accordance with claim 10, further comprising;
(d) sizing said plasmid-lipid particles to achieve a uniform particle size.
14. A method in accordance with claim 10, wherein said cationic lipids are selected from the group consisting of DODAC, DDAB, DOTAP, DOTMA, DOSPA, DOGS, DC-Chol and combinations thereof.
15. A method in accordance with claim 10, wherein said non-cationic lipids are selected from the group consisting of DOPE, POPC, EPC and combinations thereof.
16. A plasmid-lipid particle prepared according to claim 1.
17. A method for introducing a plasmid into a cell, comprising;
(a) preparing a plasmid-lipid particle according to the method of claim 1; and
(b) contacting said cell with said plasmid-lipid particle for a period of time sufficient to introduce said plasmid into said cell.
18. A method in accordance with claim 17, wherein said plasmid-lipid particle comprises a plasmid, DODAC, POPC and a PEG-Ceramide selected from the group consisting of PEG-Cer-C20 and PEG-Cer-C14.
19. A method in accordance with claim 17, wherein said plasmid-lipid particle comprises a plasmid, DODAC, DOPE and a PEG-Ceramide selected from the group consisting of PEG-Cer-C20, and PEG-Cer-C14.
20. A plasmid-lipid particle prepared according to claim 10.
21. A method for introducing a plasmid into a cell, comprising;
(a) preparing a plasmid-lipid particle according to the method of claim 10; and
(b) contacting said cell with said plasmid-lipid particle for a period of time sufficient to introduce said plasmid into said cell.
22. A method in accordance with claim 21, wherein said plasmid-lipid particle comprises a plasmid, DODAC, POPC and a PEG-Ceramide selected from the group consisting of PEG-Cer-C20 and PEG-Cer-C14.
23. A method in accordance with claim 21, wherein said plasmid-lipid particle comprises a plasmid, DODAC, DOPE and a PEG-Ceramide selected from the group consisting of PEG-Cer-C20 and PEG-Cer-C14.
US10/956,425 1995-06-07 2004-10-01 Methods for encapsulating plasmids in lipid bilayers Abandoned US20050079212A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US08/484,282 US5981501A (en) 1995-06-07 1995-06-07 Methods for encapsulating plasmids in lipid bilayers
US09/436,933 US6534484B1 (en) 1995-06-07 1999-11-08 Methods for encapsulating plasmids in lipid bilayers
US10/374,673 US6815432B2 (en) 1995-06-07 2003-02-24 Methods for encapsulating plasmids in lipid bilayers
US10/956,425 US20050079212A1 (en) 1995-06-07 2004-10-01 Methods for encapsulating plasmids in lipid bilayers

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10/956,425 US20050079212A1 (en) 1995-06-07 2004-10-01 Methods for encapsulating plasmids in lipid bilayers
US12/404,837 US20100041152A1 (en) 1995-06-07 2009-03-16 Methods for encapsulating plasmids in lipid bilayers

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US10/374,673 Continuation US6815432B2 (en) 1995-06-07 2003-02-24 Methods for encapsulating plasmids in lipid bilayers

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/404,837 Continuation US20100041152A1 (en) 1995-06-07 2009-03-16 Methods for encapsulating plasmids in lipid bilayers

Publications (1)

Publication Number Publication Date
US20050079212A1 true US20050079212A1 (en) 2005-04-14

Family

ID=23923500

Family Applications (5)

Application Number Title Priority Date Filing Date
US08/484,282 Expired - Lifetime US5981501A (en) 1995-06-07 1995-06-07 Methods for encapsulating plasmids in lipid bilayers
US09/436,933 Expired - Lifetime US6534484B1 (en) 1995-06-07 1999-11-08 Methods for encapsulating plasmids in lipid bilayers
US10/374,673 Expired - Fee Related US6815432B2 (en) 1995-06-07 2003-02-24 Methods for encapsulating plasmids in lipid bilayers
US10/956,425 Abandoned US20050079212A1 (en) 1995-06-07 2004-10-01 Methods for encapsulating plasmids in lipid bilayers
US12/404,837 Abandoned US20100041152A1 (en) 1995-06-07 2009-03-16 Methods for encapsulating plasmids in lipid bilayers

Family Applications Before (3)

Application Number Title Priority Date Filing Date
US08/484,282 Expired - Lifetime US5981501A (en) 1995-06-07 1995-06-07 Methods for encapsulating plasmids in lipid bilayers
US09/436,933 Expired - Lifetime US6534484B1 (en) 1995-06-07 1999-11-08 Methods for encapsulating plasmids in lipid bilayers
US10/374,673 Expired - Fee Related US6815432B2 (en) 1995-06-07 2003-02-24 Methods for encapsulating plasmids in lipid bilayers

Family Applications After (1)

Application Number Title Priority Date Filing Date
US12/404,837 Abandoned US20100041152A1 (en) 1995-06-07 2009-03-16 Methods for encapsulating plasmids in lipid bilayers

Country Status (1)

Country Link
US (5) US5981501A (en)

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030125292A1 (en) * 2001-11-07 2003-07-03 Sean Semple Mucoscal vaccine and methods for using the same
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
US20050191342A1 (en) * 2003-10-11 2005-09-01 Inex Pharmaceuticals Corporation Methods and compositions for enhancing innate immunity and antibody dependent cellular cytotoxicity
US20080200417A1 (en) * 1997-05-14 2008-08-21 The University Of British Columbia High efficiency encapsulation of charged therapeutic agents in lipid vesicles
WO2010107955A2 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF BTB AND CNC HOMOLOGY 1, BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR 1 (BACH 1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA) SEQUENCE LISTING
WO2010107952A2 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF CONNECTIVE TISSUE GROWTH FACTOR (CTGF) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010107957A2 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF GATA BINDING PROTEIN 3 (GATA3) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010107958A1 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 6 (STAT6) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111471A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1 (STAT1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111468A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF THE NERVE GROWTH FACTOR BETA CHAIN (NGFß) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (SINA)
WO2010111490A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF THE THYMIC STROMAL LYMPHOPOIETIN (TSLP) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111464A1 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF APOPTOSIS SIGNAL-REGULATING KINASE 1 (ASK1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111497A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF THE INTERCELLULAR ADHESION MOLECULE 1 (ICAM-1)GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2012018754A2 (en) 2010-08-02 2012-02-09 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF CATENIN (CADHERIN-ASSOCIATED PROTEIN), BETA 1 (CTNNB1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2012027206A1 (en) 2010-08-24 2012-03-01 Merck Sharp & Dohme Corp. SINGLE-STRANDED RNAi AGENTS CONTAINING AN INTERNAL, NON-NUCLEIC ACID SPACER
WO2012027467A1 (en) 2010-08-26 2012-03-01 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF PROLYL HYDROXYLASE DOMAIN 2 (PHD2) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2012058210A1 (en) 2010-10-29 2012-05-03 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACIDS (siNA)
US9181321B2 (en) 2013-03-14 2015-11-10 Shire Human Genetic Therapies, Inc. CFTR mRNA compositions and related methods and uses
US9308281B2 (en) 2011-06-08 2016-04-12 Shire Human Genetic Therapies, Inc. MRNA therapy for Fabry disease
US9522176B2 (en) 2013-10-22 2016-12-20 Shire Human Genetic Therapies, Inc. MRNA therapy for phenylketonuria
US9850269B2 (en) 2014-04-25 2017-12-26 Translate Bio, Inc. Methods for purification of messenger RNA
US9957499B2 (en) 2013-03-14 2018-05-01 Translate Bio, Inc. Methods for purification of messenger RNA

Families Citing this family (190)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030026782A1 (en) * 1995-02-07 2003-02-06 Arthur M. Krieg Immunomodulatory oligonucleotides
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6008202A (en) 1995-01-23 1999-12-28 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US5795587A (en) 1995-01-23 1998-08-18 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US5981501A (en) * 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US7422902B1 (en) * 1995-06-07 2008-09-09 The University Of British Columbia Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US7285288B1 (en) 1997-10-03 2007-10-23 Board Of Regents, The University Of Texas System Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US6977244B2 (en) * 1996-10-04 2005-12-20 Board Of Regents, The University Of Texas Systems Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US7704962B1 (en) * 1997-10-03 2010-04-27 Board Of Regents, The University Of Texas System Small oligonucleotides with anti-tumor activity
US6884430B1 (en) * 1997-02-10 2005-04-26 Aventis Pharma S.A. Formulation of stabilized cationic transfection agent(s) /nucleic acid particles
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US20050249794A1 (en) * 1999-08-27 2005-11-10 Semple Sean C Compositions for stimulating cytokine secretion and inducing an immune response
US20030104044A1 (en) * 1997-05-14 2003-06-05 Semple Sean C. Compositions for stimulating cytokine secretion and inducing an immune response
US6734171B1 (en) 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
US20040247662A1 (en) * 1998-06-25 2004-12-09 Dow Steven W. Systemic immune activation method using nucleic acid-lipid complexes
US6693086B1 (en) * 1998-06-25 2004-02-17 National Jewish Medical And Research Center Systemic immune activation method using nucleic acid-lipid complexes
US20030022854A1 (en) 1998-06-25 2003-01-30 Dow Steven W. Vaccines using nucleic acid-lipid complexes
US7189705B2 (en) 2000-04-20 2007-03-13 The University Of British Columbia Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
US6852334B1 (en) * 1999-04-20 2005-02-08 The University Of British Columbia Cationic peg-lipids and methods of use
US7094423B1 (en) * 1999-07-15 2006-08-22 Inex Pharmaceuticals Corp. Methods for preparation of lipid-encapsulated therapeutic agents
FI20000421A0 (en) * 2000-02-23 2000-02-23 Arto Urtti transport of the nucleotides-containing compounds useful in the 1,4-dihydropyridine derivatives,
ES2336766T3 (en) * 2000-10-04 2010-04-16 Kyowa Hakko Kirin Co., Ltd. Method for coating fine particles with lipid film.
US20110301569A1 (en) 2001-01-20 2011-12-08 Gordon Wayne Dyer Methods and apparatus for the CVCS
DE10109897A1 (en) * 2001-02-21 2002-11-07 Novosom Ag Optional cationic liposomes and the use of this
US7858117B2 (en) * 2002-02-21 2010-12-28 Novosom Ag Amphoteric liposomes and their use
US20030077829A1 (en) * 2001-04-30 2003-04-24 Protiva Biotherapeutics Inc.. Lipid-based formulations
EP1383480A4 (en) * 2001-04-30 2006-05-24 Targeted Genetics Corp Lipid-comprising drug delivery complexes and methods for their production
DK1479985T3 (en) 2002-01-17 2017-09-25 Alfa Laval Corp Ab Submerged evaporator comprising a plate heat exchanger and a cylindrical housing in which the plate heat exchanger is located
US20040013649A1 (en) * 2002-05-10 2004-01-22 Inex Pharmaceuticals Corporation Cancer vaccines and methods of using the same
JP4699025B2 (en) 2002-05-15 2011-06-08 サッター・ウエスト・ベイ・ホスピタルズSutter West Bay Hospitals Delivery of a nucleic-acid-like compound
EP2823809B1 (en) 2002-06-28 2016-11-02 Protiva Biotherapeutics Inc. Method and apparatus for producing liposomes
CA2502015A1 (en) 2002-12-11 2004-06-24 Coley Pharmaceutical Group, Inc. 5' cpg nucleic acids and methods of use
US20070178066A1 (en) * 2003-04-21 2007-08-02 Hall Frederick L Pathotropic targeted gene delivery system for cancer and other disorders
EP1619951B1 (en) 2003-04-21 2011-06-22 Epeius Biotechnologies Corporation Methods and compositions for treating disorders
US20050013812A1 (en) * 2003-07-14 2005-01-20 Dow Steven W. Vaccines using pattern recognition receptor-ligand:lipid complexes
AU2004257373B2 (en) 2003-07-16 2011-03-24 Arbutus Biopharma Corporation Lipid encapsulated interfering RNA
EP2338995A3 (en) * 2003-08-28 2012-01-11 Novartis AG Interfering RNA duplex having blunt-ends and 3'-modifications
CA2551022C (en) * 2003-09-15 2013-06-04 Protiva Biotherapeutics, Inc. Polyethyleneglycol-modified lipid compounds and uses thereof
US20050181035A1 (en) * 2004-02-17 2005-08-18 Dow Steven W. Systemic immune activation method using non CpG nucleic acids
JP4937899B2 (en) 2004-03-12 2012-05-23 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. iRNA agents targeting VEGF
US20050260260A1 (en) * 2004-05-19 2005-11-24 Edward Kisak Liposome compositions for the delivery of macromolecules
EP2471921A1 (en) 2004-05-28 2012-07-04 Asuragen, Inc. Methods and compositions involving microRNA
CA2569645C (en) 2004-06-07 2014-10-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use
CA2569664C (en) 2004-06-07 2013-07-16 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering rna
CA2572439A1 (en) * 2004-07-02 2006-01-12 Protiva Biotherapeutics, Inc. Immunostimulatory sirna molecules and uses therefor
WO2006007712A1 (en) * 2004-07-19 2006-01-26 Protiva Biotherapeutics, Inc. Methods comprising polyethylene glycol-lipid conjugates for delivery of therapeutic agents
CA2857879A1 (en) 2004-11-12 2006-12-28 Asuragen, Inc. Methods and compositions involving mirna and mirna inhibitor molecules
EP2199298A1 (en) * 2004-11-17 2010-06-23 Protiva Biotherapeutics Inc. Sirna silencing of Apolipoprotein B
WO2006077857A1 (en) * 2005-01-18 2006-07-27 National University Corporation Hokkaido University Method for coating particle with lipid film
JP2008536874A (en) * 2005-04-15 2008-09-11 ボード オブ リージェンツ ザ ユニバーシティー オブ テキサス システム Delivery of siRNA by the neutral lipid composition
WO2007012191A1 (en) * 2005-07-27 2007-02-01 Protiva Biotherapeutics, Inc. Systems and methods for manufacturing liposomes
US20070054873A1 (en) * 2005-08-26 2007-03-08 Protiva Biotherapeutics, Inc. Glucocorticoid modulation of nucleic acid-mediated immune stimulation
US7838658B2 (en) * 2005-10-20 2010-11-23 Ian Maclachlan siRNA silencing of filovirus gene expression
US8101741B2 (en) 2005-11-02 2012-01-24 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
GB0608838D0 (en) 2006-05-04 2006-06-14 Novartis Ag Organic compounds
US8598333B2 (en) * 2006-05-26 2013-12-03 Alnylam Pharmaceuticals, Inc. SiRNA silencing of genes expressed in cancer
US7915399B2 (en) 2006-06-09 2011-03-29 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
EP2057179A4 (en) * 2006-08-24 2010-11-10 British Columbia Cancer Agency Compositions and methods for treating myelosuppression
AU2007299804A1 (en) * 2006-09-19 2008-03-27 Asuragen, Inc. MiR-200 regulated genes and pathways as targets for therapeutic intervention
US9273300B2 (en) * 2007-02-07 2016-03-01 Strike Bio, Inc Methods and compositions for modulating sialic acid production and treating hereditary inclusion body myopathy
JP5475643B2 (en) * 2007-05-04 2014-04-16 マリーナ バイオテック,インコーポレイテッド Amino acid lipids and its use
CA2733676A1 (en) 2007-08-10 2009-02-19 British Columbia Cancer Agency Branch Microrna compositions and methods for the treatment of myelogenous leukemia
EP2198050A1 (en) 2007-09-14 2010-06-23 Asuragen, INC. Micrornas differentially expressed in cervical cancer and uses thereof
CN104672311A (en) * 2007-10-02 2015-06-03 玛瑞纳生物技术有限公司 Lipopeptides for delivery of nucleic acids
EP2207903A4 (en) * 2007-11-09 2012-02-15 Univ Northeastern Self-assembling micelle-like nanoparticles for systemic gene delivery
US20090130017A1 (en) * 2007-11-19 2009-05-21 Searete Llc Targeted short-lived drug delivery
US8071562B2 (en) 2007-12-01 2011-12-06 Mirna Therapeutics, Inc. MiR-124 regulated genes and pathways as targets for therapeutic intervention
EP2617828B1 (en) 2007-12-10 2014-09-24 Alnylam Pharmaceuticals Inc. Compositions and methods for inhibiting expression of factor VII gene
EP2238251B1 (en) 2007-12-27 2015-02-11 Protiva Biotherapeutics Inc. Silencing of polo-like kinase expression using interfering rna
AU2009213147A1 (en) 2008-02-11 2009-08-20 Rxi Pharmaceuticals Corp. Modified RNAi polynucleotides and uses thereof
EA019531B1 (en) 2008-03-05 2014-04-30 Элнилэм Фармасьютикалз, Инк. COMPOSITIONS AND METHODS FOR INHIBITING GENE EXPRESSION Eg5 and VEGF
CA2721333A1 (en) 2008-04-15 2009-10-22 Protiva Biotherapeutics, Inc. Novel lipid formulations for nucleic acid delivery
EP2770057A1 (en) 2008-04-15 2014-08-27 Protiva Biotherapeutics Inc. Silencing of CSN5 gene expression using interfering RNA
WO2009137807A2 (en) 2008-05-08 2009-11-12 Asuragen, Inc. Compositions and methods related to mirna modulation of neovascularization or angiogenesis
WO2010008582A2 (en) 2008-07-18 2010-01-21 Rxi Pharmaceuticals Corporation Phagocytic cell drug delivery system
WO2010028054A1 (en) 2008-09-02 2010-03-11 Alnylam Europe Ag. Compositions and methods for inhibiting expression of mutant egfr gene
US8664189B2 (en) 2008-09-22 2014-03-04 Rxi Pharmaceuticals Corporation RNA interference in skin indications
JP5529142B2 (en) 2008-09-25 2014-06-25 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. Lipid formulations compositions and methods for inhibiting the expression of serum amyloid a gene
ES2475065T3 (en) 2008-10-09 2014-07-10 Tekmira Pharmaceuticals Corporation Aminolpidos improved and methods for delivery of nucleic acids
EP2902013A1 (en) * 2008-10-16 2015-08-05 Marina Biotech, Inc. Processes and Compositions for Liposomal and Efficient Delivery of Gene Silencing Therapeutics
EA201792626A1 (en) 2008-10-20 2018-08-31 Элнилэм Фармасьютикалз, Инк. Compositions and methods for inhibiting transtiretin expression
WO2010059226A2 (en) 2008-11-19 2010-05-27 Rxi Pharmaceuticals Corporation Inhibition of map4k4 through rnai
CA2746514C (en) 2008-12-10 2018-11-27 Alnylam Pharmaceuticals, Inc. Gnaq targeted dsrna compositions and methods for inhibiting expression
WO2010078536A1 (en) 2009-01-05 2010-07-08 Rxi Pharmaceuticals Corporation Inhibition of pcsk9 through rnai
US9745574B2 (en) 2009-02-04 2017-08-29 Rxi Pharmaceuticals Corporation RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
US20120041051A1 (en) 2009-02-26 2012-02-16 Kevin Fitzgerald Compositions And Methods For Inhibiting Expression Of MIG-12 Gene
CN104922699A (en) 2009-03-12 2015-09-23 阿尔尼拉姆医药品有限公司 LIPID FORMULATED COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Eg5 AND VEGF GENES
US8569256B2 (en) 2009-07-01 2013-10-29 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
EP2449114B9 (en) 2009-07-01 2017-04-19 Protiva Biotherapeutics Inc. Novel lipid formulations for delivery of therapeutic agents to solid tumors
US9018187B2 (en) 2009-07-01 2015-04-28 Protiva Biotherapeutics, Inc. Cationic lipids and methods for the delivery of therapeutic agents
WO2011011447A1 (en) 2009-07-20 2011-01-27 Protiva Biotherapeutics, Inc. Compositions and methods for silencing ebola virus gene expression
EP2810643A3 (en) 2009-08-14 2015-03-11 Alnylam Pharmaceuticals Inc. Lipid formulated compositions and mehods for inhibiting expression of a gene from the ebola virus
CA2775092A1 (en) 2009-09-23 2011-03-31 Protiva Biotherapeutics, Inc. Compositions and methods for silencing genes expressed in cancer
WO2011056682A1 (en) 2009-10-27 2011-05-12 The University Of British Columbia Reverse head group lipids, lipid particle compositions comprising reverse headgroup lipids, and methods for the delivery of nucleic acids
RU2573409C2 (en) 2009-11-04 2016-01-20 Дзе Юниверсити Оф Бритиш Коламбиа Lipid particles containing nucleic acids and related methods
NZ700688A (en) 2009-12-01 2016-02-26 Shire Human Genetic Therapies Delivery of mrna for the augmentation of proteins and enzymes in human genetic diseases
CN103200945B (en) 2010-03-24 2016-07-06 雷克西制药公司 rna interference in ocular symptoms
RU2615143C2 (en) 2010-03-24 2017-04-04 Адвирна Self-delivered rnai compounds of reduced size
CN103108642B (en) 2010-03-24 2015-09-23 雷克西制药公司 rna interference with skin fibrosis symptoms in
CA2794307A1 (en) 2010-03-26 2011-09-29 Mersana Therapeutics, Inc. Modified polymers for delivery of polynucleotides, method of manufacture, and methods of use thereof
CA2792291A1 (en) 2010-03-29 2011-10-06 Kumamoto University Sirna therapy for transthyretin (ttr) related ocular amyloidosis
WO2011133529A1 (en) * 2010-04-19 2011-10-27 The University Of North Carolina At Chapel Hill Predictors of pharmacokinetic and pharmacodynamic disposition of carrier-mediated agents
EP2382994A1 (en) 2010-04-26 2011-11-02 Maurizio Victor Cattaneo Ligand targeted nanocapsules for the delivery of RNAi and other agents
US20130156845A1 (en) 2010-04-29 2013-06-20 Alnylam Pharmaceuticals, Inc. Lipid formulated single stranded rna
US10077232B2 (en) 2010-05-12 2018-09-18 Arbutus Biopharma Corporation Cyclic cationic lipids and methods of use
US20130123338A1 (en) 2010-05-12 2013-05-16 Protiva Biotherapeutics, Inc. Novel cationic lipids and methods of use thereof
CA2801066A1 (en) 2010-06-02 2011-12-08 Alnylam Pharmaceuticals, Inc. Compositions and methods directed to treating liver fibrosis
WO2011160062A2 (en) 2010-06-17 2011-12-22 The Usa As Represented By The Secretary, National Institutes Of Health Compositions and methods for treating inflammatory conditions
WO2012000104A1 (en) 2010-06-30 2012-01-05 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US9339513B2 (en) 2010-11-09 2016-05-17 Alnylam Pharmaceuticals, Inc. Lipid formulated compositions and methods for inhibiting expression of Eg5 and VEGF genes
WO2012075040A2 (en) 2010-11-30 2012-06-07 Shire Human Genetic Therapies, Inc. mRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES
EP2648763A4 (en) 2010-12-10 2014-05-14 Alnylam Pharmaceuticals Inc Compositions and methods for inhibiting expression of klf-1 and bcl11a genes
US20140212434A1 (en) 2011-02-03 2014-07-31 The United States of America, as represented by the Secretary,Department of Health & HumanServices Multivalent vaccines for rabies virus and filoviruses
US10196637B2 (en) 2011-06-08 2019-02-05 Nitto Denko Corporation Retinoid-lipid drug carrier
US9011903B2 (en) 2011-06-08 2015-04-21 Nitto Denko Corporation Cationic lipids for therapeutic agent delivery formulations
MX344807B (en) 2011-06-21 2017-01-09 Alnylam Pharmaceuticals Inc Compositions and methods for inhibition of expression of apolipoprotein c-iii (apoc3) genes.
EP2723351B1 (en) 2011-06-21 2018-02-14 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibition of expression of protein c (proc) genes
WO2012177784A2 (en) 2011-06-21 2012-12-27 Alnylam Pharmaceuticals Angiopoietin-like 3 (angptl3) irna compostions and methods of use thereof
WO2012178033A2 (en) 2011-06-23 2012-12-27 Alnylam Pharmaceuticals, Inc. Serpina1 sirnas: compositions of matter and methods of treatment
WO2013019857A2 (en) 2011-08-01 2013-02-07 Alnylam Pharmaceuticals, Inc. Method for improving the success rate of hematopoietic stem cell transplants
US9644241B2 (en) 2011-09-13 2017-05-09 Interpace Diagnostics, Llc Methods and compositions involving miR-135B for distinguishing pancreatic cancer from benign pancreatic disease
CA2853316C (en) 2011-10-25 2018-11-27 The University Of British Columbia Limit size lipid nanoparticles and related methods
US9579338B2 (en) 2011-11-04 2017-02-28 Nitto Denko Corporation Method of producing lipid nanoparticles for drug delivery
WO2013093648A2 (en) 2011-11-04 2013-06-27 Nitto Denko Corporation Method of producing lipid nanoparticles for drug delivery
US9035039B2 (en) 2011-12-22 2015-05-19 Protiva Biotherapeutics, Inc. Compositions and methods for silencing SMAD4
NZ700075A (en) 2012-02-24 2016-05-27 Protiva Biotherapeutics Inc Trialkyl cationic lipids and methods of use thereof
US9133461B2 (en) 2012-04-10 2015-09-15 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the ALAS1 gene
US9127274B2 (en) 2012-04-26 2015-09-08 Alnylam Pharmaceuticals, Inc. Serpinc1 iRNA compositions and methods of use thereof
CA2876155A1 (en) 2012-06-08 2013-12-12 Ethris Gmbh Pulmonary delivery of mrna to non-lung target cells
JP2016506240A (en) 2012-12-05 2016-03-03 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. PCSK9iRNA compositions and methods of use thereof
CN105555954B (en) 2013-03-14 2019-03-26 吉恩维沃公司 Improved thymidine kinase gene
AU2014239184B2 (en) 2013-03-14 2018-11-08 Translate Bio, Inc. Methods and compositions for delivering mRNA coded antibodies
SG11201507400SA (en) 2013-03-14 2015-10-29 Alnylam Pharmaceuticals Inc Complement component c5 irna compositions and methods of use thereof
US10342760B2 (en) 2013-03-15 2019-07-09 The University Of British Columbia Lipid nanoparticles for transfection and related methods
ES2670529T3 (en) 2013-03-15 2018-05-30 Translate Bio, Inc. Synergistic enhancement of the delivery of nucleic acids by mixed formulations
KR20140119513A (en) 2013-04-01 2014-10-10 삼성전자주식회사 Composition for nucleic acid delivery containing hyaluronic acid
RU2015154738A (en) 2013-05-22 2017-06-27 Элнилэм Фармасьютикалз, Инк. COMPOSITION FOR iRNA TMPRSS6 BASIS AND METHODS OF THEIR USE
DK2999785T3 (en) 2013-05-22 2018-07-23 Alnylam Pharmaceuticals Inc Serpina1-iRNA compositions and methods of use thereof
EP3013958A4 (en) 2013-06-25 2017-02-22 The U.S.A. as represented by the Secretary, Department of Health and Human Services Glucan-encapsulated sirna for treating type 2 diabetes mellitus
TW201534578A (en) 2013-07-08 2015-09-16 Daiichi Sankyo Co Ltd Novel lipid
WO2015050990A1 (en) 2013-10-02 2015-04-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the lect2 gene
JP6525435B2 (en) 2013-10-22 2019-06-12 シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド Lipid formulations for the delivery of messenger RNA
KR101713886B1 (en) 2013-12-19 2017-03-10 연세대학교 산학협력단 Therapeutic PRK2-silencing siRNA for the treatment of hepatitis C virus infection
CA2938857A1 (en) 2014-02-11 2015-08-20 Alnylam Pharmaceuticals, Inc. Ketohexokinase (khk) irna compositions and methods of use thereof
BR112016026950A2 (en) 2014-05-22 2017-10-31 Alnylam Pharmaceuticals Inc RNAi compositions angiotensinogen (AGT), and methods of use thereof
WO2015184256A2 (en) 2014-05-30 2015-12-03 Shire Human Genetic Therapies, Inc. Biodegradable lipids for delivery of nucleic acids
CN106795142A (en) 2014-06-24 2017-05-31 夏尔人类遗传性治疗公司 Stereochemically enriched compositions for delivery of nucleic acids
AU2015280499A1 (en) 2014-06-25 2017-02-09 Acuitas Therapeutics Inc. Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
AU2015283954A1 (en) 2014-07-02 2016-12-22 Translate Bio, Inc. Encapsulation of messenger RNA
EP3191591A1 (en) 2014-09-12 2017-07-19 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting complement component c5 and methods of use thereof
EP3201338A1 (en) 2014-10-02 2017-08-09 Protiva Biotherapeutics, Inc. Compositions and methods for silencing hepatitis b virus gene expression
WO2016061487A1 (en) 2014-10-17 2016-04-21 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting aminolevulinic acid synthase-1 (alas1) and uses thereof
EP3212794A2 (en) 2014-10-30 2017-09-06 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting serpinc1 (at3) and methods of use thereof
BR112017008868A2 (en) 2014-11-10 2018-01-16 Alnylam Pharmaceuticals Inc IRNA compositions of the hepatitis B virus (HBV) and methods for their use
WO2016081444A1 (en) 2014-11-17 2016-05-26 Alnylam Pharmaceuticals, Inc. Apolipoprotein c3 (apoc3) irna compositions and methods of use thereof
EP3226912A1 (en) 2014-12-05 2017-10-11 Rana Therapeutics Inc. Messenger rna therapy for treatment of articular disease
WO2016130806A2 (en) 2015-02-13 2016-08-18 Alnylam Pharmaceuticals, Inc. Patatin-like phospholipase domain containing 3 (pnpla3) irna compositions and methods of use thereof
EP3270894A1 (en) 2015-03-19 2018-01-24 Translate Bio, Inc. Mrna therapy for pompe disease
US20180135057A1 (en) 2015-04-08 2018-05-17 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the lect2 gene
EP3292201A2 (en) 2015-05-06 2018-03-14 Alnylam Pharmaceuticals, Inc. Factor xii (hageman factor) (f12), kallikrein b, plasma (fletcher factor) 1 (klkb1), and kininogen 1 (kng1) irna compositions and methods of use thereof
US20160346219A1 (en) 2015-06-01 2016-12-01 Autotelic Llc Phospholipid-coated therapeutic agent nanoparticles and related methods
WO2016197132A1 (en) 2015-06-04 2016-12-08 Protiva Biotherapeutics Inc. Treating hepatitis b virus infection using crispr
WO2016201301A1 (en) 2015-06-12 2016-12-15 Alnylam Pharmaceuticals, Inc. Complement component c5 irna compositions and methods of use thereof
EP3310918A1 (en) 2015-06-18 2018-04-25 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting hydroxyacid oxidase (glycolate oxidase, hao1) and methods of use thereof
WO2016209862A1 (en) 2015-06-23 2016-12-29 Alnylam Pharmaceuticals, Inc. Glucokinase (gck) irna compositions and methods of use thereof
AU2016285852A1 (en) 2015-06-29 2017-12-21 Acuitas Therapeutics Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US20180201929A1 (en) 2015-07-10 2018-07-19 Alnylam Pharmaceuticals, Inc. INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN, ACID LABILE SUBUNIT (IGFALS) AND INSULIN-LIKE GROWTH FACTOR 1 (IGF-1) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
US20180208932A1 (en) 2015-07-29 2018-07-26 Arbutus Biopharma Corporation Compositions and methods for silencing hepatitis b virus gene expression
CN108368507A (en) 2015-09-02 2018-08-03 阿尔尼拉姆医药品有限公司 PROGRAMMED CELL DEATH 1 LIGAND 1 (PD-L1) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
WO2017048843A1 (en) 2015-09-14 2017-03-23 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the alas1 gene
WO2017048620A1 (en) 2015-09-14 2017-03-23 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting patatin-like phospholipase domain containing 3 (pnpla3) and methods of use thereof
EP3362555A1 (en) 2015-10-14 2018-08-22 Translate Bio, Inc. Modification of rna-related enzymes for enhanced production
EP3368507A1 (en) 2015-10-28 2018-09-05 Acuitas Therapeutics, Inc. Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
WO2017100542A1 (en) 2015-12-10 2017-06-15 Alnylam Pharmaceuticals, Inc. Sterol regulatory element binding protein (srebp) chaperone (scap) irna compositions and methods of use thereof
EP3439695A1 (en) 2016-04-04 2019-02-13 The U.S.A. as represented by the Secretary, Department of Health and Human Services Multivalent vaccines for rabies virus and coronaviruses
WO2017177169A1 (en) 2016-04-08 2017-10-12 Rana Therapeutics, Inc. Multimeric coding nucleic acid and uses thereof
WO2017184689A1 (en) 2016-04-19 2017-10-26 Alnylam Pharmaceuticals, Inc. High density lipoprotein binding protein (hdlbp/vigilin) irna compositions and methods of use thereof
WO2017214518A1 (en) 2016-06-10 2017-12-14 Alnylam Pharmaceuticals, Inc. COMPLETMENT COMPONENT C5 iRNA COMPOSTIONS AND METHODS OF USE THEREOF FOR TREATING PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)
CA3034681A1 (en) 2016-06-30 2018-01-04 Arbutus Biopharma Corporation Compositions and methods for delivering messenger rna
UY37376A (en) 2016-08-26 2018-03-23 Amgen Inc RNAi constructs to inhibit expression of asgr1 and methods for their use
TW201831690A (en) 2016-10-31 2018-09-01 美商艾歐凡斯生物治療公司 For amplification of tumor infiltrating lymphocytes engineered artificial antigen presenting cell
TW201829443A (en) 2016-11-23 2018-08-16 美商阿尼拉製藥公司 Serine proteinase inhibitor factor A1 iRNA composition and method of use
WO2019103857A1 (en) 2017-11-22 2019-05-31 Iovance Biotherapeutics, Inc. Expansion of peripheral blood lymphocytes (pbls) from peripheral blood
WO2019089922A1 (en) 2017-11-01 2019-05-09 Alnylam Pharmaceuticals, Inc. Complement component c3 irna compositions and methods of use thereof
WO2019099610A1 (en) 2017-11-16 2019-05-23 Alnylam Pharmaceuticals, Inc. Kisspeptin 1 (kiss1) irna compositions and methods of use thereof
WO2019100023A1 (en) 2017-11-17 2019-05-23 Iovance Biotherapeutics, Inc. Til expansion from fine needle aspirates and small biopsies
WO2019118638A2 (en) 2017-12-12 2019-06-20 Amgen Inc. Rnai constructs for inhibiting pnpla3 expression and methods of use thereof
WO2019126097A1 (en) 2017-12-18 2019-06-27 Alnylam Pharmaceuticals, Inc. High mobility group box-1 (hmgb1) irna compositions and methods of use thereof
WO2019136459A1 (en) 2018-01-08 2019-07-11 Iovance Biotherapeutics, Inc. Processes for generating til products enriched for tumor antigen-specific t-cells
WO2019136456A1 (en) 2018-01-08 2019-07-11 Iovance Biotherapeutics, Inc. Processes for generating til products enriched for tumor antigen-specific t-cells

Citations (64)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4394448A (en) * 1978-02-24 1983-07-19 Szoka Jr Francis C Method of inserting DNA into living cells
US4438052A (en) * 1980-01-16 1984-03-20 Hans Georg Weder Process and device for producing bilayer vesicles
US4515736A (en) * 1983-05-12 1985-05-07 The Regents Of The University Of California Method for encapsulating materials into liposomes
US4598051A (en) * 1980-03-12 1986-07-01 The Regents Of The University Of California Liposome conjugates and diagnostic methods therewith
US4897355A (en) * 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5013556A (en) * 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5171678A (en) * 1989-04-17 1992-12-15 Centre National De La Recherche Scientifique Lipopolyamines, their preparation and their use
US5208036A (en) * 1985-01-07 1993-05-04 Syntex (U.S.A.) Inc. N-(ω, (ω-1)-dialkyloxy)- and N-(ω, (ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5225212A (en) * 1989-10-20 1993-07-06 Liposome Technology, Inc. Microreservoir liposome composition and method
US5262168A (en) * 1987-05-22 1993-11-16 The Liposome Company, Inc. Prostaglandin-lipid formulations
US5264618A (en) * 1990-04-19 1993-11-23 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
US5279833A (en) * 1990-04-04 1994-01-18 Yale University Liposomal transfection of nucleic acids into animal cells
US5283185A (en) * 1991-08-28 1994-02-01 University Of Tennessee Research Corporation Method for delivering nucleic acids into cells
US5320906A (en) * 1986-12-15 1994-06-14 Vestar, Inc. Delivery vehicles with amphiphile-associated active ingredient
US5545412A (en) * 1985-01-07 1996-08-13 Syntex (U.S.A.) Inc. N-[1, (1-1)-dialkyloxy]-and N-[1, (1-1)-dialkenyloxy]-alk-1-yl-n,n,n-tetrasubstituted ammonium lipids and uses therefor
US5703055A (en) * 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
US5705385A (en) * 1995-06-07 1998-01-06 Inex Pharmaceuticals Corporation Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5785992A (en) * 1994-09-30 1998-07-28 Inex Pharmaceuticals Corp. Compositions for the introduction of polyanionic materials into cells
US5820873A (en) * 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
US5835395A (en) * 1991-02-07 1998-11-10 Texas Instruments Incorporated Eprom pinout option
US5885613A (en) * 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US5965542A (en) * 1997-03-18 1999-10-12 Inex Pharmaceuticals Corp. Use of temperature to control the size of cationic liposome/plasmid DNA complexes
US5976567A (en) * 1995-06-07 1999-11-02 Inex Pharmaceuticals Corp. Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5981501A (en) * 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US6086913A (en) * 1995-11-01 2000-07-11 University Of British Columbia Liposomal delivery of AAV vectors
US6093816A (en) * 1996-06-27 2000-07-25 Isis Pharmaceuticals, Inc. Cationic lipids
US6110745A (en) * 1997-07-24 2000-08-29 Inex Pharmaceuticals Corp. Preparation of lipid-nucleic acid particles using a solvent extraction and direct hydration method
US6143276A (en) * 1997-03-21 2000-11-07 Imarx Pharmaceutical Corp. Methods for delivering bioactive agents to regions of elevated temperatures
US6143716A (en) * 1996-10-15 2000-11-07 The Liposome Company, Inc. Liposomal peptide-lipid conjugates and delivery using same
US6194388B1 (en) * 1994-07-15 2001-02-27 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6287591B1 (en) * 1997-05-14 2001-09-11 Inex Pharmaceuticals Corp. Charged therapeutic agents encapsulated in lipid particles containing four lipid components
US6320017B1 (en) * 1997-12-23 2001-11-20 Inex Pharmaceuticals Corp. Polyamide oligomers
US6339068B1 (en) * 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US6355267B1 (en) * 1993-11-05 2002-03-12 Amgen Inc. Liposome preparation and material encapsulation method
US6365179B1 (en) * 1999-04-23 2002-04-02 Alza Corporation Conjugate having a cleavable linkage for use in a liposome
US6406705B1 (en) * 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US6410328B1 (en) * 1998-02-03 2002-06-25 Protiva Biotherapeutics Inc. Sensitizing cells to compounds using lipid-mediated gene and compound delivery
US6417326B1 (en) * 1996-04-11 2002-07-09 The University Of British Columbia Fusogenic liposomes
US6447800B2 (en) * 1996-01-18 2002-09-10 The University Of British Columbia Method of loading preformed liposomes using ethanol
US20030035829A1 (en) * 1997-07-24 2003-02-20 Townsend And Townsend And Crew Liposomal compositions for the delivery of nucleic acid catalysts
US20030050268A1 (en) * 2001-03-29 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases
US20030104044A1 (en) * 1997-05-14 2003-06-05 Semple Sean C. Compositions for stimulating cytokine secretion and inducing an immune response
US6585224B1 (en) * 2000-08-01 2003-07-01 Bombardier Motor Corporation Of America Outboard motor rack system and related method of use
US6586410B1 (en) * 1995-06-07 2003-07-01 Inex Pharmaceuticals Corporation Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US20030125292A1 (en) * 2001-11-07 2003-07-03 Sean Semple Mucoscal vaccine and methods for using the same
US6627616B2 (en) * 1995-12-13 2003-09-30 Mirus Corporation Intravascular delivery of non-viral nucleic acid
US20030212026A1 (en) * 1999-09-25 2003-11-13 University Of Iowa Research Foundation Immunostimulatory nucleic acids
US6673364B1 (en) * 1995-06-07 2004-01-06 The University Of British Columbia Liposome having an exchangeable component
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
US20040009943A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Pathogen vaccines and methods for using the same
US20040013649A1 (en) * 2002-05-10 2004-01-22 Inex Pharmaceuticals Corporation Cancer vaccines and methods of using the same
US20040023649A1 (en) * 2000-08-08 2004-02-05 Torsten Bing Method for conducting data communications with subscriber stations, and radio communications network for implementing said method
US6734171B1 (en) * 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
US20040266719A1 (en) * 1998-05-22 2004-12-30 Mccluskie Michael J. Methods and products for inducing mucosal immunity
US6841537B1 (en) * 1998-04-22 2005-01-11 Protiva Biotherapeutics Inc. Combination therapy using nucleic acids and conventional drugs
US20050059619A1 (en) * 2002-08-19 2005-03-17 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US20050130911A1 (en) * 2003-09-25 2005-06-16 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
US20050191342A1 (en) * 2003-10-11 2005-09-01 Inex Pharmaceuticals Corporation Methods and compositions for enhancing innate immunity and antibody dependent cellular cytotoxicity
US6949520B1 (en) * 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon
US20050249794A1 (en) * 1999-08-27 2005-11-10 Semple Sean C Compositions for stimulating cytokine secretion and inducing an immune response
US7094423B1 (en) * 1999-07-15 2006-08-22 Inex Pharmaceuticals Corp. Methods for preparation of lipid-encapsulated therapeutic agents
US20060255153A1 (en) * 2005-05-13 2006-11-16 Lite-On Semiconductor Corp. Handheld fingerprint-capturing device
US7223887B2 (en) * 2001-12-18 2007-05-29 The University Of British Columbia Multivalent cationic lipids and methods of using same in the production of lipid particles

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5858784A (en) * 1991-12-17 1999-01-12 The Regents Of The University Of California Expression of cloned genes in the lung by aerosol- and liposome-based delivery
WO1995002698A1 (en) * 1993-07-12 1995-01-26 Life Technologies, Inc. Composition and methods for transfecting eukaryotic cells
US5753613A (en) * 1994-09-30 1998-05-19 Inex Pharmaceuticals Corporation Compositions for the introduction of polyanionic materials into cells
JP2000516948A (en) * 1996-08-26 2000-12-19 トランジェーヌ、ソシエテ、アノニム Cationic lipid - nucleic acid complexes
US6835395B1 (en) * 1997-05-14 2004-12-28 The University Of British Columbia Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles
US6852334B1 (en) * 1999-04-20 2005-02-08 The University Of British Columbia Cationic peg-lipids and methods of use

Patent Citations (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4394448A (en) * 1978-02-24 1983-07-19 Szoka Jr Francis C Method of inserting DNA into living cells
US4438052A (en) * 1980-01-16 1984-03-20 Hans Georg Weder Process and device for producing bilayer vesicles
US4598051A (en) * 1980-03-12 1986-07-01 The Regents Of The University Of California Liposome conjugates and diagnostic methods therewith
US4515736A (en) * 1983-05-12 1985-05-07 The Regents Of The University Of California Method for encapsulating materials into liposomes
US4897355A (en) * 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5545412A (en) * 1985-01-07 1996-08-13 Syntex (U.S.A.) Inc. N-[1, (1-1)-dialkyloxy]-and N-[1, (1-1)-dialkenyloxy]-alk-1-yl-n,n,n-tetrasubstituted ammonium lipids and uses therefor
US5208036A (en) * 1985-01-07 1993-05-04 Syntex (U.S.A.) Inc. N-(ω, (ω-1)-dialkyloxy)- and N-(ω, (ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5320906A (en) * 1986-12-15 1994-06-14 Vestar, Inc. Delivery vehicles with amphiphile-associated active ingredient
US5262168A (en) * 1987-05-22 1993-11-16 The Liposome Company, Inc. Prostaglandin-lipid formulations
US5703055A (en) * 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
US5171678A (en) * 1989-04-17 1992-12-15 Centre National De La Recherche Scientifique Lipopolyamines, their preparation and their use
US5013556A (en) * 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5225212A (en) * 1989-10-20 1993-07-06 Liposome Technology, Inc. Microreservoir liposome composition and method
US5279833A (en) * 1990-04-04 1994-01-18 Yale University Liposomal transfection of nucleic acids into animal cells
US5264618A (en) * 1990-04-19 1993-11-23 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
US5835395A (en) * 1991-02-07 1998-11-10 Texas Instruments Incorporated Eprom pinout option
US5283185A (en) * 1991-08-28 1994-02-01 University Of Tennessee Research Corporation Method for delivering nucleic acids into cells
US6355267B1 (en) * 1993-11-05 2002-03-12 Amgen Inc. Liposome preparation and material encapsulation method
US6194388B1 (en) * 1994-07-15 2001-02-27 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US5785992A (en) * 1994-09-30 1998-07-28 Inex Pharmaceuticals Corp. Compositions for the introduction of polyanionic materials into cells
US5885613A (en) * 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US5820873A (en) * 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
US20070172950A1 (en) * 1995-06-07 2007-07-26 The University Of British Columbia Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use of gene transfer
US5705385A (en) * 1995-06-07 1998-01-06 Inex Pharmaceuticals Corporation Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US6534484B1 (en) * 1995-06-07 2003-03-18 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US6815432B2 (en) * 1995-06-07 2004-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US6673364B1 (en) * 1995-06-07 2004-01-06 The University Of British Columbia Liposome having an exchangeable component
US6586410B1 (en) * 1995-06-07 2003-07-01 Inex Pharmaceuticals Corporation Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5976567A (en) * 1995-06-07 1999-11-02 Inex Pharmaceuticals Corp. Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5981501A (en) * 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US6086913A (en) * 1995-11-01 2000-07-11 University Of British Columbia Liposomal delivery of AAV vectors
US6627616B2 (en) * 1995-12-13 2003-09-30 Mirus Corporation Intravascular delivery of non-viral nucleic acid
US6447800B2 (en) * 1996-01-18 2002-09-10 The University Of British Columbia Method of loading preformed liposomes using ethanol
US6417326B1 (en) * 1996-04-11 2002-07-09 The University Of British Columbia Fusogenic liposomes
US6093816A (en) * 1996-06-27 2000-07-25 Isis Pharmaceuticals, Inc. Cationic lipids
US6143716A (en) * 1996-10-15 2000-11-07 The Liposome Company, Inc. Liposomal peptide-lipid conjugates and delivery using same
US6406705B1 (en) * 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US5965542A (en) * 1997-03-18 1999-10-12 Inex Pharmaceuticals Corp. Use of temperature to control the size of cationic liposome/plasmid DNA complexes
US6143276A (en) * 1997-03-21 2000-11-07 Imarx Pharmaceutical Corp. Methods for delivering bioactive agents to regions of elevated temperatures
US7341738B2 (en) * 1997-05-14 2008-03-11 The University Of British Columbia Lipid-encapsulated polyanionic nucleic acid
US6287591B1 (en) * 1997-05-14 2001-09-11 Inex Pharmaceuticals Corp. Charged therapeutic agents encapsulated in lipid particles containing four lipid components
US20050008689A1 (en) * 1997-05-14 2005-01-13 Inex Pharmaceuticals Corporation High efficiency encapsulation of charged therapeutic agents in lipid vesicles
US20030129221A1 (en) * 1997-05-14 2003-07-10 Semple Sean C. High efficiency encapsulation of charged therapeutic agents in lipid vesicles
US20030104044A1 (en) * 1997-05-14 2003-06-05 Semple Sean C. Compositions for stimulating cytokine secretion and inducing an immune response
US6858225B2 (en) * 1997-05-14 2005-02-22 Inex Pharmaceuticals Corporation Lipid-encapsulated polyanionic nucleic acid
US6339068B1 (en) * 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US20030035829A1 (en) * 1997-07-24 2003-02-20 Townsend And Townsend And Crew Liposomal compositions for the delivery of nucleic acid catalysts
US6110745A (en) * 1997-07-24 2000-08-29 Inex Pharmaceuticals Corp. Preparation of lipid-nucleic acid particles using a solvent extraction and direct hydration method
US6734171B1 (en) * 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
US6586559B2 (en) * 1997-12-23 2003-07-01 Inex Pharmaceuticals Corporation Polyamide oligomers
US6320017B1 (en) * 1997-12-23 2001-11-20 Inex Pharmaceuticals Corp. Polyamide oligomers
US6410328B1 (en) * 1998-02-03 2002-06-25 Protiva Biotherapeutics Inc. Sensitizing cells to compounds using lipid-mediated gene and compound delivery
US6841537B1 (en) * 1998-04-22 2005-01-11 Protiva Biotherapeutics Inc. Combination therapy using nucleic acids and conventional drugs
US20040266719A1 (en) * 1998-05-22 2004-12-30 Mccluskie Michael J. Methods and products for inducing mucosal immunity
US6365179B1 (en) * 1999-04-23 2002-04-02 Alza Corporation Conjugate having a cleavable linkage for use in a liposome
US7094423B1 (en) * 1999-07-15 2006-08-22 Inex Pharmaceuticals Corp. Methods for preparation of lipid-encapsulated therapeutic agents
US20060257465A1 (en) * 1999-07-15 2006-11-16 The University Of British Columbia Methods for preparation of lipid-encapsulated therapeutic agents
US20050249794A1 (en) * 1999-08-27 2005-11-10 Semple Sean C Compositions for stimulating cytokine secretion and inducing an immune response
US20030212026A1 (en) * 1999-09-25 2003-11-13 University Of Iowa Research Foundation Immunostimulatory nucleic acids
US7271156B2 (en) * 1999-09-25 2007-09-18 University Of Iowa Research Foundation Immunostimulatory nucleic acids
US6949520B1 (en) * 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon
US6585224B1 (en) * 2000-08-01 2003-07-01 Bombardier Motor Corporation Of America Outboard motor rack system and related method of use
US20040023649A1 (en) * 2000-08-08 2004-02-05 Torsten Bing Method for conducting data communications with subscriber stations, and radio communications network for implementing said method
US20030050268A1 (en) * 2001-03-29 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases
US20030125292A1 (en) * 2001-11-07 2003-07-03 Sean Semple Mucoscal vaccine and methods for using the same
US7223887B2 (en) * 2001-12-18 2007-05-29 The University Of British Columbia Multivalent cationic lipids and methods of using same in the production of lipid particles
US20040009943A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Pathogen vaccines and methods for using the same
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
US20040013649A1 (en) * 2002-05-10 2004-01-22 Inex Pharmaceuticals Corporation Cancer vaccines and methods of using the same
US20050059619A1 (en) * 2002-08-19 2005-03-17 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US20050130911A1 (en) * 2003-09-25 2005-06-16 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
US20050191342A1 (en) * 2003-10-11 2005-09-01 Inex Pharmaceuticals Corporation Methods and compositions for enhancing innate immunity and antibody dependent cellular cytotoxicity
US20060255153A1 (en) * 2005-05-13 2006-11-16 Lite-On Semiconductor Corp. Handheld fingerprint-capturing device

Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8021686B2 (en) 1997-05-14 2011-09-20 The University Of British Columbia Lipid-encapsulated polyanionic nucleic acid
US20080200417A1 (en) * 1997-05-14 2008-08-21 The University Of British Columbia High efficiency encapsulation of charged therapeutic agents in lipid vesicles
US20030125292A1 (en) * 2001-11-07 2003-07-03 Sean Semple Mucoscal vaccine and methods for using the same
US20040009944A1 (en) * 2002-05-10 2004-01-15 Inex Pharmaceuticals Corporation Methylated immunostimulatory oligonucleotides and methods of using the same
US20050191342A1 (en) * 2003-10-11 2005-09-01 Inex Pharmaceuticals Corporation Methods and compositions for enhancing innate immunity and antibody dependent cellular cytotoxicity
WO2010107955A2 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF BTB AND CNC HOMOLOGY 1, BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR 1 (BACH 1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA) SEQUENCE LISTING
WO2010107952A2 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF CONNECTIVE TISSUE GROWTH FACTOR (CTGF) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010107957A2 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF GATA BINDING PROTEIN 3 (GATA3) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010107958A1 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 6 (STAT6) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111471A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1 (STAT1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111490A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF THE THYMIC STROMAL LYMPHOPOIETIN (TSLP) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111464A1 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF APOPTOSIS SIGNAL-REGULATING KINASE 1 (ASK1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111497A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF THE INTERCELLULAR ADHESION MOLECULE 1 (ICAM-1)GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2010111468A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF THE NERVE GROWTH FACTOR BETA CHAIN (NGFß) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (SINA)
WO2012018754A2 (en) 2010-08-02 2012-02-09 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF CATENIN (CADHERIN-ASSOCIATED PROTEIN), BETA 1 (CTNNB1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
EP3372684A1 (en) 2010-08-24 2018-09-12 Sirna Therapeutics, Inc. Single-stranded rnai agents containing an internal, non-nucleic acid spacer
WO2012027206A1 (en) 2010-08-24 2012-03-01 Merck Sharp & Dohme Corp. SINGLE-STRANDED RNAi AGENTS CONTAINING AN INTERNAL, NON-NUCLEIC ACID SPACER
WO2012027467A1 (en) 2010-08-26 2012-03-01 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF PROLYL HYDROXYLASE DOMAIN 2 (PHD2) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2012058210A1 (en) 2010-10-29 2012-05-03 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACIDS (siNA)
EP3327125A1 (en) 2010-10-29 2018-05-30 Sirna Therapeutics, Inc. Rna interference mediated inhibition of gene expression using short interfering nucleic acids (sina)
US10238754B2 (en) 2011-06-08 2019-03-26 Translate Bio, Inc. Lipid nanoparticle compositions and methods for MRNA delivery
US9308281B2 (en) 2011-06-08 2016-04-12 Shire Human Genetic Therapies, Inc. MRNA therapy for Fabry disease
US9597413B2 (en) 2011-06-08 2017-03-21 Shire Human Genetic Therapies, Inc. Pulmonary delivery of mRNA
US10350303B1 (en) 2011-06-08 2019-07-16 Translate Bio, Inc. Lipid nanoparticle compositions and methods for mRNA delivery
US9957499B2 (en) 2013-03-14 2018-05-01 Translate Bio, Inc. Methods for purification of messenger RNA
US9713626B2 (en) 2013-03-14 2017-07-25 Rana Therapeutics, Inc. CFTR mRNA compositions and related methods and uses
US9181321B2 (en) 2013-03-14 2015-11-10 Shire Human Genetic Therapies, Inc. CFTR mRNA compositions and related methods and uses
US9522176B2 (en) 2013-10-22 2016-12-20 Shire Human Genetic Therapies, Inc. MRNA therapy for phenylketonuria
US10208295B2 (en) 2013-10-22 2019-02-19 Translate Bio, Inc. MRNA therapy for phenylketonuria
US10155785B2 (en) 2014-04-25 2018-12-18 Translate Bio, Inc. Methods for purification of messenger RNA
US9850269B2 (en) 2014-04-25 2017-12-26 Translate Bio, Inc. Methods for purification of messenger RNA

Also Published As

Publication number Publication date
US6534484B1 (en) 2003-03-18
US6815432B2 (en) 2004-11-09
US20100041152A1 (en) 2010-02-18
US5981501A (en) 1999-11-09
US20030181410A1 (en) 2003-09-25

Similar Documents

Publication Publication Date Title
Hung et al. Nonviral vectors for gene therapy
Mahato et al. Biodistribution and gene expression of lipid/plasmid complexes after systemic administration
US6071533A (en) Preparation of stable formulations of lipid-nucleic acid complexes for efficient in vivo delivery
Karanth et al. pH‐Sensitive liposomes‐principle and application in cancer therapy
Montier et al. Progress in cationic lipid-mediated gene transfection: a series of bio-inspired lipids as an example
CN1082043C (en) Novel cationic lipids and the use thereof
JP5587352B2 (en) Material Blood - Artificial for transporting across the brain barrier low density lipoprotein carrier
CN1882693B (en) Polyethyleneglycol-modified lipid compounds and uses thereof
EP1781593B1 (en) Cationic lipids and methods of use
Templeton et al. Improved DNA: liposome complexes for increased systemic delivery and gene expression
JP5480764B2 (en) Amphoteric liposomes and their use
US6656498B1 (en) Cationic liposomes for gene transfer
AU2003237864B2 (en) Delivery of nucleic acid-like compounds
Niculescu-Duvaz et al. Structure-activity relationship in cationic lipid mediated gene transfection
de Lima et al. Cationic liposomes for gene delivery: from biophysics to biological applications
DE69631149T2 (en) Formulations in the form of emulsions for administration of nucleic acids to cells
Kaneda Virosomes: evolution of the liposome as a targeted drug delivery system
EP0708637B1 (en) Self-assembling polynucleotide delivery system comprising dendrimer polycations
AU2004257373B2 (en) Lipid encapsulated interfering RNA
Tranchant et al. Physicochemical optimisation of plasmid delivery by cationic lipids
Bhattacharya et al. Advances in gene delivery through molecular design of cationic lipids
US5990089A (en) Self-assembling polynucleotide delivery system comprising dendrimer polycations
US6410328B1 (en) Sensitizing cells to compounds using lipid-mediated gene and compound delivery
US6120798A (en) Liposome-entrapped polynucleotide composition and method
JP3887015B2 (en) Novel compositions for the introduction of polyanionic material into cells

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE UNIVERSITY OF BRITISH COLUMBIA, CANADA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:INEX PHARMACEUTICALS CORPORATION;REEL/FRAME:018731/0706

Effective date: 20070108

AS Assignment

Owner name: TEKMIRA PHARMACEUTICALS CORPORATION, CANADA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:INEX PHARMACEUTICALS CORPORATION;REEL/FRAME:019287/0631

Effective date: 20070430

Owner name: THE UNIVERSITY OF BRITISH COLUMBIA, CANADA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:INEX PHARMACEUTICALS CORPORATION;REEL/FRAME:019287/0631

Effective date: 20070430

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: SILICON VALLEY BANK, CALIFORNIA

Free format text: SECURITY AGREEMENT;ASSIGNOR:TEKMIRA PHARMACEUTICALS CORPORATION;REEL/FRAME:027464/0745

Effective date: 20111221