|Publication number||US20050079212 A1|
|Application number||US 10/956,425|
|Publication date||Apr 14, 2005|
|Filing date||Oct 1, 2004|
|Priority date||Jun 7, 1995|
|Also published as||US5981501, US6534484, US6815432, US20030181410, US20100041152|
|Publication number||10956425, 956425, US 2005/0079212 A1, US 2005/079212 A1, US 20050079212 A1, US 20050079212A1, US 2005079212 A1, US 2005079212A1, US-A1-20050079212, US-A1-2005079212, US2005/0079212A1, US2005/079212A1, US20050079212 A1, US20050079212A1, US2005079212 A1, US2005079212A1|
|Inventors||Jeffery Wheeler, Michael Hope, Pieter Cullis, Marcel Bally|
|Original Assignee||Inex Pharmaceuticals Corp.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (74), Referenced by (11), Classifications (12), Legal Events (3)|
|External Links: USPTO, USPTO Assignment, Espacenet|
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.
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.
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.
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
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.
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
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:
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;
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., M
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, H
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.
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.
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
Calculation of % DODAC in vesicles for any given DODAC:DNA charge ratio
as a function of total mg of lipid
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)
Dialysis Method (Particles with POPC:DODAC:PEG-Cer(C20)
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.
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
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
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.
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
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
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
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.
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
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.
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).
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.
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|WO2010111468A2||Mar 25, 2010||Sep 30, 2010||Merck Sharp & Dohme Corp.||RNA INTERFERENCE MEDIATED INHIBITION OF THE NERVE GROWTH FACTOR BETA CHAIN (NGFß) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (SINA)|
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|WO2010111490A2||Mar 25, 2010||Sep 30, 2010||Merck Sharp & Dohme Corp.||RNA INTERFERENCE MEDIATED INHIBITION OF THE THYMIC STROMAL LYMPHOPOIETIN (TSLP) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)|
|WO2010111497A2||Mar 25, 2010||Sep 30, 2010||Merck Sharp & Dohme Corp.||RNA INTERFERENCE MEDIATED INHIBITION OF THE INTERCELLULAR ADHESION MOLECULE 1 (ICAM-1)GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)|
|U.S. Classification||424/450, 435/458|
|International Classification||C12N15/88, A61K9/127|
|Cooperative Classification||Y10S514/851, Y10S436/829, Y10S977/907, Y10S977/80, C12N15/88, A61K9/1272|
|European Classification||A61K9/127B2, C12N15/88|
|Jan 10, 2007||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
|May 15, 2007||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
|Dec 29, 2011||AS||Assignment|
Owner name: SILICON VALLEY BANK, CALIFORNIA
Free format text: SECURITY AGREEMENT;ASSIGNOR:TEKMIRA PHARMACEUTICALS CORPORATION;REEL/FRAME:027464/0745
Effective date: 20111221