Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
  • A61K9/1272—Non-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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• Plasmid DNA-cationic liposome complexes are currently the most commonly employed nonviral gene delivery vehicles (Feigner, Scientific American 276:102-106 (1997); Chonn, et al, Current Opinion in Biotechnology 6:698-708 (1995)).
• complexes are large, poorly defined systems that are not suited for systemic applications and can elicit considerable toxic side effects (Harrison, et al, Biotechniques 19:816-823 (1995); Huang, et al, Nature Biotechnology 15:620-621 (1997); Templeton, et al, Nature Biotechnology 15:647-652 (1997); Hofland, et al, Pharmaceutical Research 14:742-749 (1997)).
• SPLP stabilized plasmid-lipid particles
• DOPE lipid dioleoylphosphatidylethanolamine
• PEG poly(ethylene glycol)
• SPLP have systemic application as they exhibit extended circulation lifetimes following intravenous (i.v.) injection, accumulate preferentially at distal tumour sites due to the enhanced vascular permeability in such regions, and can me iate transgene expression at these tumour sites.
• T e levels of transgene expression observed at the tumour site following i.v. injection of SPLP containing the luciferase marker gene are superior to the levels that can be achieved employing plasmid DNA-cationic liposome complexes (lipoplexes) or naked DNA. Still, improved levels of expression may be required for optimal therapeutic benefit in some applications (see, e.g., Monck, et al, J. Drug
• Cationic poly(ethylene glycol) (PEG) lipids have been designed for insertion into lipid bilayers to impart a positive charge ⁇ ee, Chen, et al, Bioconj. Chem.
• CPL containing distearoyl-PE (DSPE) coupled to PEG containing one or more distal positive charges were synthesized, and shown to promote enhanced in vitro cellular binding and uptake of liposomes (Chen, etal, Bioconj. Chem.
• the present invention provides effective compositions, methods and uses for delivering nucleic acids to cells.
• the inventive compositions and methods are based upon the surprising discovery that the presence of an endosomal membrane destabilizer in a lipid formulation leads to a dramatic increase in transfection efficiency.
• the present compositions and methods can be used in vitro or in vivo, and can be used to increase the transfection efficiency of any cell type, including mammalian cells (e.g. , human).
• the present invention provides a nucleic acid- lipid particle composition for introducing a nucleic acid into a cell comprising: a cationic lipid, a conjugated lipid that inhibits aggregation of particles, a nucleic acid and an endosomal membrane destabilizer.
• the nucleic acid-lipid particles are "stabilized plasmid-lipid particles" (SPLP).
• SPLP stabilized plasmid-lipid particles
• SPLP are less than 150 nm in diameter and comprise a single plasmid encapsulated within a bilayer lipid vesicle.
• the conjugated lipid that inhibits aggregation typically comprises a hydrophilic polymer.
• the hydrophilic polymer is a PEG or poly amide (e.g., ATT A) having a molecular weight of about 250 to about 7000 daltons.
• the endosomal membrane destabilizer can be inside the particle, outside the particle, or both inside and outside the particle.
• the endosomal membrane destabilizer is Ca ion.
• the concentration of Ca '1" * " ion is from about 0.1 mM to about 100 mM.
• the conjugated lipid that inhibits aggregation is a "cationic polymer lipid” (CPL).
• CPL cationic polymer lipid
• the CPL has the formula
• A is a lipid moiety
• W is a hydrophilic polymer
• Y is a polycationic moiety
• Y is selected from lysine, arginine, asparagine, glutamine, and combinations thereof.
• the present invention provides a method for introducing a nucleic acid into a cell, comprising contacting the cell with a nucleic acid-lipid particle composition, wherein the particle comprises a cationic lipid, a conjugated lipid that inhibits aggregation of particles, a nucleic acid; and an endosomal membrane destabilizer.
• the endosomal membrane destabilizer can be inside the particle, outside the particle, or both inside and outside the particle. In certain embodiments, the endosomal membrane destabilizer contacts the cell before the particle, after the particle, simultaneously or combinations thereof.
• the present invention provides a method for inducing H ⁇ phase structure in a lipid bilayer, comprising contacting the lipid bilayer with an endosomal membrane destabilizer, thereby inducing H ⁇ phase structure in the lipid bilayer.
• the endosomal membrane destabilizer e.g., Ca "1 " ion
• compositions, methods and uses offer numerous advantages.
• the presence of an endosomal membrane destabilizer leads to a dramatic increase in the transfection efficiency of nucleic acids.
• transfection efficiency the amount of gene product within the cell is greatly increased.
• present compositions and methods can be used in vitro or in vivo, and can be used to increase the transfection efficiency of any cell type, including human.
• Figure 1 Effect and specificity of Ca 2+ on SPLP transfection. Increasing concentrations of CaCl 2 ( • ), MgCl 2 ( ⁇ ), or NaCl ( ⁇ ) (0 to 14 mM) were titrated into SPLP prior to their addition to cells. 0.5 ⁇ g of pCMVLuc plasmid encapsulated in SPLP (DODAC/DOPE/PEG-CerC20; 7:83:10 mol/mol/mol) vesicles was used to transfect cells plated at 1 x 10 4 cells/well of 96-well plates. Cells were incubated with SPLP for 24 h, and Luc activity was measured as described in Materials and Methods, Exampl I. All experiments were performed in triplicate.
• Figure 2 Effect of Ca 2+ on the cellular uptake of SPLP.
• SPLP containing 0.5 mol% Rd-labeled DOPE (DODAC/DOPE/PEG-CerC20/Rh-DOPE; 7:82.5:10:0.5 mol/mol/mol/mol) were employed to monitor cellular lipid uptake.
• 80 nmoles of lipid vesicles prepared in the presence of Ca 2+ (0 to 14 mM) were incubated on cells until the appropriate time periods.
• Levels of lipid uptake were determined by measuring Rd fluorescence at 4 h ( • ), 8 h ( ⁇ ), or 24 h ( ⁇ *> ) as described in Materials and Methods, Example I. All experiments were performed in triplicate.
• FIG. 3 Fluorescence micrographs of the cellular localization of SPLP. 100 nmoles of 4 mol% Rh-DOPE labeled vesicles were incubated on cells (plated at lxlO 5 cells per well of a 12- well plate) in the absence (A) or presence (B) of calcium (10 mM). At 8 h time point, transfecting media was replaced with complete DMEM media and cells were examined using fluorescence microscopy.
• FIG. 4 Intracellular processing of plasmid DNA was affected by the presence of Ca 2+ .
• SPLP containing 2.5 ⁇ g plasmid DNA was used to transfect BHK cells in the absence ( • ) or presence ( ⁇ ) of 8 mM Ca 2+ as described in Materials and Methods, Example I.
• DNA was extracted from the cells and intracellular DNA was detected by hybridization to a specific 32 P-labeled plasmid DNA probe.
• A Levels of plasmid DNA uptake determined by dot blot analysis as described in Materials and Methods.
• B Integrity of intracellular plasmid DNA determined by Southern blot analysis.
• Lanes 1 and 11 pCMVLuc control; lanes 2, 5, 8 and 12: untransfected control; lanes 3, 6, 9 and 13: cells transfected with SPLP; lanes 4, 7, 10 and 14: cells rans ec e wi an m a ; an ane : ce s rans ec e wi an m Mg 2+ . All experiments were performed in triplicate.
• FIG. 5 P NMR spectra of various model membrane systems in the presence of CaCl 2 .
• Ca 2+ was titrated into the vesicles (DOPE/DOPS/DOPC/Chol, 1 :1:1:3) at Ca 2+ /DOPS ratios ranging from 0: 1 to 0.5: 1 (molar ratios).
• Ca + was titrated into the vesicles (DOPE/DOPS/DOPC/Chol/DODAC, 1:1:1:3:0.25) at Ca 2+ /DOPS ratios ranging from 0:1 to 0.25:1 (molar ratios). Equilibration of the cations across the lipid bilayers was ensured by three cycles of freeze-thawing. Spectra have been scaled to the same peak height. Experiments were carried out as described in Materials and Methods, Example I.
• FIG. 6 Effect of Ca 2+ -containing SPLP on transfection.
• Ca 2+ was loaded employing A23187 in the presence of a pH gradient as described in Materials and Methods. Increasing concentrations of Ca 2+ (0 to 14 mM) were added to both SPLP ( ⁇ ) and Ca 2+ - containing SPLP (•) prior to DMEM dilution.
• 0.5 ⁇ g of pCMVLuc plasmid encapsulated in SPLP were used to transfect cells plated at 1 x 10 4 cells/well of 96-well plates. Luc activity was measured as described in Materials and Methods, Example I. All experiments were performed in triplicate.
• Figure 7 Effect of Ca 2+ on improved SPLP systems.
• SPLP containing higher DODAC content (14 mol%) or CPL (4 mol%) were used to transfection cells in the presence (dashed bars) or absence (open bars) of 8 mM Ca 2+ .
• 0.5 ⁇ g of pCMVLuc was used in each formulation in each transfection experiment.
• Cells were exposed to the vesicles for 24 h before assaying for Luc expression, as outlined in Materials and Methods, Example I. Experiments were performed in triplicate.
• FIG. 8 Production of SPLP-CPL 4 .
• CPL 4 possesses four positive charges at the end of a PEG 34 oo molecule attached to a lipid achor, DSPE.
• Dansylated CPL 4 (0.3 ⁇ mol) was added to SPLP composed of 6 ⁇ mol DOPE:PEG- CerC 20 :DODAC:Rh-PE (83.5:10:6:0.5; mol%) containing 360 ⁇ g pCMVLuc in a total volume of 1.5 mL and incubated at 60°C. Aliquots (250 ⁇ L) of the mixture were taken at the times indicated and unincorporated CPL was removed employing Sepharose CL-4B column c roma ograp y. incorpora ion was e ermine as escri e in a eria s an e o s, Example II.
• FIG 11. Freeze-fracture electron micrographs of (A) SPLP, (B) SPLP- CPL 4 and (C) SPLP-CPL 4 in the presence of 40 mM CaCl 2 .
• the SPLP-CPL 4 were prepared as described in Materials and Methods, Example II, and contained 4 mol% CPL .
• the bar in plate A corresponds to 200 nm.
• Figure 12. Serum stability of SPLP-CPL as assayed by Southern analysis of encapsulated plasmid. SPLP were prepared as indicated in the legend to Figure 9 and 4 mol% of CPL 4 inserted using the post-insertion protocol.
• SPLP-CPL containing 5 ⁇ g pCMVLuc were incubated in the presence of 50% mouse serum at 37°C for the times indicated, an aliquot of the mixture corresponding to 1 ⁇ g of plasmid DNA was removed and plasmid DNA was extracted and subjected to Southern analysis, as described in the Materials and Methods.
• Lanes 1-4 indicate the behaviour of naked plasmid DNA following 0, 1, 2, and 4 h incubation times respectively; lanes 5-8 indicate the behaviour of plasmid extracted from SPLP following 0, 1, 2, and 4 h incubation times; and lanes 9-12 show the behaviour of plasmid DNA extracted from SPLP containing 4 mol% CPL 4 following 0, 1, 2, and 4 h incubation times.
• FIG 13 A Influence of the amount of CPL incorporated into SPLP on the uptake of SPLP-CPL 4 into BHK cells.
• the insertion of CPL into SPLP and the preparation of lipoplexes was performed as described in Materials and Methods, Example II.
• the SPLP-CPL 4 media contained 40 mM CaCl 2 to prevent aggregation, addition to the BHK cells resulted in dilution of the CaCl 2 concentration to 8 mM.
• the uptake protocol involved incubation of SPLP-CPL 4 (20 ⁇ M total lipid) with 10 5 BHK cells in DMEM containing 10% FBS. Following incu a on, e ce s were yse an upta e o r o am ne- was measure as escr e n Materials and Methods, Example II.
• FIG. 14 Luciferase expression in BHK cells following transfection by SPLP containing various amounts of CPL .
• SPLP containing 2, 3 and 4 mol% CPL 4 were prepared employing the post-insertion process.
• BHK cells (10 4 ) were transfected with SPLP, SPLP-CPL 4 and DOPE:DODAC (1:1) lipoplexes containing 5.0 ⁇ g/mL pCMVLuc using a transfection time of 4 h and a complete incubation time of 24 h, as described in Materials and Methods, Example II.
• the CaCl 2 concentration in the SPLP-CPL -containing systems following dilution with media and addition to the BHK cells was 8 mM. After transfection the cells were lysed and the luciferase and BCA assays performed as described in Materials and Methods.
• SPLP-CLP 4 SPLP-CPL containing 4 mol% CPL 4 were prepared by the post-insertion process as described in Materials and Methods, Example II. Increasing concentrations of CaCl 2 or MgCl 2 were added to the SPLP-CPL 4 (5.0 ⁇ g pCMVLuc/mL), transferred to BHK cells and incubated for 48 h in DMEM containing 10% FBS. The cells were then lysed and the luciferase activity and protein content were measured as described in Materials and Methods, Example II.
• FIG. 16 Effect of Ca 2+ (•) and Mg 2+ ( ⁇ ) on the uptake of SPLP-CPL 4 by BHK cells.
• SPLP-CPL were prepared with increasing cation concentrations as indicated for Figure 8 and incubated with BHK cells (-80 ⁇ M lipid and ⁇ 5.0 ⁇ g pCMVLuc/mL per well) for 4 h in DMEM containing 10% FBS. The cells were then lysed and the SPLP-CPL 4 content (as indicated by the Rh-PE lipid label) and cellular protein measured as described in Materials and Methods, Example II.
• FIG. 1 Luciferase expression in BHK cells as a function of transfection time for SPLP, SPLP-CPL 4 and lipoplexes.
• SPLP-CPL 4 containing 4 mol% CPL 4 were prepared by the post-insertion process.
• BHK cells in DMEM and 10% FBS were incubated with SPLP, SPLP-CPL and lipoplexes (5.0 ⁇ g/mL pCMVLuc) employing transfection times of 4, 8 and 24 h and total incubation times of 24 h.
• the final CaCl 2 concentration following addition of media was 8 mM.
• the cells were then assayed for luciferase activity and protein con ent.
• Lipofectin lipoplexes ( ⁇ ) is plotted as a function of transfection time. Lipoplexes were prepared at a charge ratio of 1.5:1.
• FIG 18A The transfection potency of SPLP-CPL (•) containing 4 mol% CPL 4 and and Lipofectin lipoplexes ( ⁇ ) following extended transfection times with BHK cells.
• SPLP-CPL 4 and lipoplexes were generated as indicated for Figure 10.
• BHK cells were transfected in DMEM containing 10% FBS for 24 and 48 h with SPLP-CPL 4 and Lipofectin lipoplexes (charge ratio of 1.5:1) containing 5.0 ⁇ g/mL pCMVLuc.
• charge ratio of 1.5:1 containing 5.0 ⁇ g/mL pCMVLuc.
• the luciferase expression levels and cell protein levels were determined in the cell lysate.
• the luciferase activity was normalized for protein content in the lysate and plotted as a function of transfection time.
• FIG. 19 Fluorescence and phase contrast micrographs of BHK cells transfected with SPLP-CPL 4 and lipoplexes containing a plasmid coding for GFP.
• Cells were transfected with SPLP-CPL 4 for 24 h (Al, A2) and 48 h (Bl, B2) and with lipofectin for 24 h (CI, C2).
• SPLP and lipoplexes were prepared with pCMVGFP as described in Materials and Methods, Example II.
• SPLP-CPL 4 containing 4 mol% CPL was prepared by the post- insertion process and contained CaCl 2 , resulting in an 8 mM CaCl concentration in the transfection medium.
• BHK cells (10 5 ) were incubated with SPLP-CPL or Lipofectin (5.0 ⁇ g/mL) in DMEM containing 10% FBS for the 24 and 48 h transfection times and examined immediately after the transfection period.
• FIG. 20 Mechanism for disruption of cellular membranes mediated by cationic lipoplexes. Following binding (Step 1) and endocytosis (Step 2) into a target cell, cationic lipoplexes are transferred to late endosomal compartments (Step 3). Cationic lipids induce destabilization of the endosomal membrane leading to fusion (Step 4) of the lipoplex with the endosomal membrane, or complete remodeling of the endosomal membrane into a nonbilayer phase (Step 5).
• Figure 21 A synthetic scheme for the preparation of cationic-PEG-lipid conjugates having varying amount of charged head groups (a.) Et 3 N/CHCl 3 ; (b.) TFA /CHC1 3 ; c. Et 3 N / CHCI 3 N ⁇ , N ⁇ -di-t-Boc-L-Lysine N-hydroxysuccinide ester. AND PREFERRED EMBODIMENTS I. Introduction
• compositions and methods for delivering nucleic acids to cells. These compositions and methods are based upon the discovery that the presence of an endosomal membrane destabilizer (e.g., calcium) leads to a dramatic increase in the transfection efficiency of nucleic acids (e.g., plasmids) formulated as SPLP or "stabilized nucleic acid (e.g., plasmid)-lipid particles.”
• an endosomal membrane destabilizer e.g., calcium
• SPLP stabilized nucleic acid
• SPLP are less than about 150 nm in diameter (more preferably about 70 nm in diameter) and consist of a single plasmid encapsulated within a bilayer lipid vesicle.
• EMD endosomal membrane destabilizer
• endosomes are typically distinct intracellular compartments isolated from the rest of the cell by a selectively permeable membrane.
• Suitable EMDs include, but are not limited to, monovalent metal ions such as K , Na , divalent metal ions such as Mg , Ca , Mn , Co , and combinations of the metal ions with cationic lipids.
• the most preferred EMD is C times higher transfection efficiency is observed for SPLPs containing Ca 2+ ions than SPLPs in the absence of Ca 2+ ions.
• the present methods can be used in vitro or in vivo, and can be used to increase the transfection efficiency of any cell type, including mammalian cells.
• an endosomal membrane destabilizer e.g., calcium
• any of a wide range of calcium concentrations can be used, ranging, for example, from 0.1 mM to 100 mM. Preferably, from about 1 mM to about 20 mM is used, most preferably from about 8 to about 10 mM.
• the endosomal membrane destabilizer e.g., calcium
• the endosomal membrane destabilizer is first added to the SPLP at a high concentration which will give rise to a desired final concentration following the dilution of the SPLP into the transfection medium.
• the endosomal membrane destabilizer e.g., calcium
• the endosomal membrane destabilizer can be co-administered with the SPLP, it can be administered prior to the administration of the SPLP or it can be administered after the administration of the SPLP.
• any method can be used that will result in a local increase of the endosomal membrane destabilizer (e.g., calcium) concentration at the site of transfection.
• the endosomal membrane destabilizer e.g., calcium
• par ic es can e ormu a e o ncorpora e e en osoma mem rane destabilizer
• particles can be soaked in a solution containing a high endosomal membrane destabilizer (e.g., calcium) concentration prior to administration, or the particles can be administered in a buffer or formulation containing a high endosomal membrane destabilizer (e.g. , calcium) concentration.
• a high endosomal membrane destabilizer e.g., calcium
• Such methods are especially useful for the local delivery of particles, e.g., intratumoral injection, where the co-administration of, e.g., calcium ions, can produce a locally high calcium concentration, thereby leading to enhanced transfection of the particles into cells at or near the site of delivery.
• the endosomal membrane destabilizer can be co-administered with the SPLP, it can be administered prior to the administration of the SPLP or it can be administered after the administration of the SPLP.
• the SPLP are formulated to include on their surface chelating molecules for chelating the endosomal membrane destabilizer, e.g., lipids derivatized with a endosomal membrane destabilizer chelator, thereby allowing the generation of a locally high endosomal membrane destabilizer concentration even following systemic delivery of the particles.
• particles are formulated to include calcium chelating molecules on the surface, e.g., lipids derivatized with a calcium chelator, thereby allowing the generation of a locally high calcium concentration even following systemic delivery of the particles.
• SPLP particle can be used to practice the present mvention.
• SPLP comprising any of a broad range of concentrations of cationic and other lipids can be used.
• the SPLP can comprise any of a wide variety of cationic and other lipids.
• the SPLP can be prepared with any plasmid, from any source and comprising any polynucleotide sequence, and can be prepared using any of a large number of methods.
• the present invention also provides SPLP containing cationic PEG lipids, called SPLP-CPL.
• SPLP-CPL 4 is used, comprising a PEG lipid having four positive charges.
• SPLP and SPLP-CPL can be derivatized to include any of a number of functional groups, including, but not limited to, calcium chelators, cell or tissue- specific targeting molecules, labels, and others.
• Suitable SPLP and SPLP-CPL for use in the present invention and methods of nialdng and using SPLP and SPLP-CPL, are taught, e.g., in U.S. Application Nos. 60/130,151 and 09/553,639, as well as in PCT International Application PCT/CA00/00451, the teachings of each of which is incorporated herein in its entirety by reference. . e ini ions
• lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids' which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; (3) “derived lipids” such as steroids.
• vesicle-forming lipid is intended to include any amphipathic lipid having a hydrophobic moiety and a polar head group, and which by itself can form spontaneously into bilayer vesicles in water, as exemplified by most phospholipids.
• vesicle-adopting lipid is intended to include any amphipathic lipid which is stably incorporated into lipid bilayers in combination with other amphipathic lipids, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.
• Vesicle-adopting lipids include lipids that on their own tend to adopt a nonlamellar phase, yet which are capable of assuming a bilayer structure in the presence of a bilayer-stabilizing component.
• a typical example is DOPE
• Bilayer stabilizing components include, but are not limited to, conjugated lipids that inhibit aggregation of the SPLPs, polyamide oligomers (e.g., ATTA-lipid derivatives), peptides, proteins, detergents, lipid-derivatives, PEG-lipid derivatives such as PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (see, U.S. Application Serial No. 08/485,608, now U.S. Patent No. 5,885,613, which is incorporated herein by reference).
• amphipathic lipid refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase.
• Amphipathic lipids are usually the major component of a lipid vesicle. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.
• Hydrophobicity can be conferred by the inclusion of apolar 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).
• apolar 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).
• amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.
• phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, pa m toy o eoy p osp a y c o ne, ysop osp at y c o ne, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine.
• amphipathic lipids Other compoimds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols and ⁇ - acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipid described above can be mixed with other lipids including triglycerides and sterols.
• neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
• lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamme, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.
• hydrophopic lipid refers to compounds having apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, l,2-diacyloxy-3-aminopropane and l,2-dialkyl-3- aminopropane.
• diacylglycerolyl denotes 2-fatty acyl chains, R and R having independently between 2 and 30 carbons bonded to the 1- and 2-position of glycerol by ester linkages.
• the acyl groups can be saturated or have varying degrees of unsaturation.
• dialkylglycerolyl denotes two C 1 -C 30 alkyl chains bonded to the 1- and 2-position of glycerol by ether linkages.
• N-N-dialkylamino denotes
• l,2-diacyloxy-3-aminopropane denotes 2-fatty acyl chains C--.-C30 bonded to the 1- and 2-position of propane by an ester linkage.
• the acyl groups can be saturated or have varying degrees of unsaturation.
• the 3 -position of the propane molecule has a -NH- group attached.
• l,2-diacyloxy-3-aminopropanes have the following general formula:
• l,2-dialkyl-3-aminopropane denotes 2-alkyl chains (C ! -C 3 o) bonded to the 1- and 2-position of propane by an ether linkage. The 3 -position of the propane molecule has a -NH- group attached. l,2-dialkyl-3-aminopropanes have the following general formula:
• noncationic lipid refers to any neutral lipid as described above as well as anionic lipids.
• anionic lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- gmtarylphosphatidylethanolamines, lysophosphatidylglycerols, and other anionic modifying groups joined to neutral lipids.
• cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.
• lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC”); N-(2,3 - dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTMA”); N,N-distearyl-N,N- dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (“DOTAP”); 3 -(N-(N',N'-dimethylaminoethane)- carbamoyl)cholesterol (“DC-Choi”) and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethy
• cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN ® (commercially available cationic liposomes comprising an , - ⁇ o eoy --.
• LIPOFECTAMLNE® commercially available cationic liposomes comprising N-(l-(2,3-dioleyloxy)propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate ("DOSPA”) and("DOPE"), from GIBCO/BRL
• TRANSFECTAM ® commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine ("DOGS”) in ethanol from Promega Corp., Madison, Wisconsin, USA).
• lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA and the like.
• fusogenic refers to the ability of a liposome, an SPLP or other drug delivery system to fuse with membranes of a cell.
• the membranes can be either the plasma membrane or membranes surrounding organelles, e.g., endosome, nucleus, etc. Fusogenesis is the fusion of a liposome to such a membrane.
• dendrimer includes reference to branched polymers that possess multiple generations. In dendrimers, each generation creates multiple branch points.
• ligand includes any molecule, compound or device with a reactive functional group and includes lipids, amphipathic lipids, carrier compounds, chelating moities, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immune stimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, targeting agents, or toxins.
• the foregoing list is illustrative and not intended to be exhaustive.
• ATT A or "polyamide” refers to, but is not limited to, compounds disclosed in U.S. Patent Application No. 09/218,988, filed December 22, 1998. These compounds include a compound having the formula
• R is a member selected from the group consisting of hydrogen, alkyl and acyl
• R 1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R 1 and the nitrogen to which they are bound form an azido moiety
• R 2 is a member of the group selected from hydrogen, optionally substituted alkyl, optionally substituted aryl and a side chain of an amino acid
• R is a member selected from the group consisting of hydrogen, a ogen, y roxy, a oxy, mercapto, y raz no, am no an , w ere n an are independently hydrogen or alkyl
• n is 4 to 80
• m is 2 to 6
• p is 1 to 4
• q is 0 or 1.
• alkyl denotes branched or unbranched hydrocarbon chains, such as, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tertbutyl, octa-decyl and 2-methylpentyl.
• These groups can be optionally substituted with one or more functional groups which are attached commonly to such chains, such as, hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano, alkylthio, heterocyclyl, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the like to form alkyl groups such as trifluoromethyl, 3- hydroxyhexyl, 2-carboxypropyl, 2-fluoroethyl, carboxymethyl, cyanobutyl and the like.
• functional groups which are attached commonly to such chains, such as, hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano, alkylthio, heterocyclyl, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl
• alkylene refers to a divalent alkyl as defined above, such as methylene (-CH 2 -), propylene (-CH 2 CH 2 CH 2 -), chloroethylene (-CHC1CH 2 -), 2-thiobutene (- CH 2 CH(SH)CH 2 CH 2 -), l-bromo-3-hydroxyl-4-methylpentene (-CHBrCH 2 CH(OH)CH(CH 3 )CH 2 -), and the like.
• alkenyl denotes branched or unbranched hydrocarbon chains containing one or more carbon-carbon double bonds.
• alkynyl refers to branched or unbranched hydrocarbon chains containing one or more carbon-carbon triple bonds.
• aryl denotes a chain of carbon atoms which form at least one aromatic ring having preferably between about 6-14 carbon atoms, such as phenyl, naphthyl, indenyl, and the like, and which may be substituted with one or more functional groups which are attached commonly to such chains, such as hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano, cyanoamido, alkylthio, heterocycle, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the like to form aryl groups such as biphenyl, iodobiphenyl, methoxybiphenyl, anthryl, bromophenyl, iodophenyl, chlorophenyl, hydroxyphenyl, methoxyphenyl, formylphenyl, acet
• acyl denotes the -C(O)R group, wherein R is alkyl or aryl as defined above, such as formyl, acetyl, propionyl, or butyryl.
• alkoxy denotes -OR-, wherein R is alkyl. e erm ami o eno es an ami e in age: - - w ere n s hydrogen or alkyl).
• amino denotes an amine linkage: -NR-, wherein R is hydrogen or alkyl or a terminal NH .
• carboxyl denotes the group -C(O)O-
• carbonyl denotes the group -C(O)-.
• carbonate indicates the group -OC(O)O-.
• carbamate denotes the group -NHC(O)O-
• urea denotes the group -NHC(O)NH-
• phosphoro denotes the group -OP(O)(OH)O-.
• basic amino acid refers to naturally-occurring amino acids as well as synthetic amino acids and/or or amino acid mimetics having a net positive charge at a selected pH, such as physiological pH. This group includes, but is not limited to, lysine, arginine, asparagine, glutamine, histidine and the like.
• phosphorylethanolamino denotes the group
• phosphorylethanolamido denotes the group -OP(O)(OH)OCH 2 CH 2 NHC(O)-.
• phospho denotes a pentavalent phosphorous moiety -P(O)(OH)O-.
• phosphoethanolamino denotes the group
• phosphoethanolamido denotes the group -P(O)(OH)OCH 2 CH 2 NHC(O)-.
• ethylene oxide unit denotes the group -OCH 2 CH 2 -.
• CPL refers to a cationic-polymer-lipid, e.g., cationic-PEG-lipid.
• Preferred CPLs are compounds of Formulae I and II. Such CPLs are disclosed in U.S. Patent Application No. 09/553,639, which was filed April 20, 2000, and PCT Patent Application No. CA 00/00451, which was filed April 20, 2000 and which published as WO 00/62813 on October 26, 2000.
• the term "d-DSPE-CPL-M" is encompassed by the term “CPL1 " which refers to a DSPE-CPL having one positive charge.
• the "d-" in d-DSPE-CPL-M indicates that the CPL contains a fluorescent dansyl group.
• d-DSPE-CPL-Tl is encompassed by the term “CPL3” which refers to DSPE-CPL having three positive charges.
• d-DSPE-CPL-Ql is encompassed by the term “CPL4a” which refers to DSPE-CPL having four positive charges.
• d-DSPE-CPL-Q5 or, alternatively, DSPE-PEGQuad5, or, alternatively, DSPE-CPL-4, are all encompassed by the term “CPL4 (or CPL4b)" which refer to a DSPE-CPL having four positive charges.
• CPL4 or CPL4b
• CPLs were synthesized which contained 1 (mono, or M), 2 (di, or D), 3 (tri, or T), and 4 (quad, or Q) positive charges.
• Various Quad CPLs were synthesized, hence these are numbered Ql through Q5.
• HBS Hepes-buffered saline
• Rho-PE rhodamine-phosphatidylethanolamine
• LUVs refers to "large unilamellar vesicles.”
• SPLPs Nucleic Acid-Lipid Particles
• the nulceic acid-lipid particles or, alternatively, SPLPs typically comprise cationic lipid and nucleic acids. Such SPLPs also preferably comprise noncationic lipid and a bilayer stabilizing component or, more preferably, a conjugated lipid that inhibits aggregation of the SPLPs.
• the SPLPs of the present invention have a mean diameter of less than about 150 nm and are substantially nontoxic.
• the nucleic acids when present in the SPLPs of the present invention are resistant to aqueous solution to degradation with a nuclease.
• Such SPLPs are disclosed in great detail in U.S. Patent No. 5,976,567 and PCT Patent Publication No. WO 96/40964, the teachings of both of which are incoporated herein by reference.
• Narious suitable cationic lipids may be used in the present invention, either alone or in combination with one or more other cationic lipid species or neutral lipid species.
• Cationic lipids which are useful in the present invention can be any of a number of lipid species which carry a net positive charge at physiological pH, for example: DODAC, DOTMA, DDAB, DOTAP, DOSPA, DOGS, DC-Choi and DMRIE, or combinations thereof.
• DODAC DODAC
• DOTMA DOTMA
• DDAB DDAB
• DOTAP DOSPA
• DOGS DOGS
• DC-Choi and DMRIE or combinations thereof.
• a number of these lipids and related analogs, which are also useful in the present invention, have been described in co-pending USS ⁇ 08/316,399; U.S. Patent ⁇ os. , , , , , , , an , , , e isc osures o w ic are incorpora e herein by reference.
• 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, New York, USA); LIP OFECT AMINE ® (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic liposomes comprising DOGS from Promega Corp., Madison, Wisconsin, USA).
• LIPOFECTIN ® commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, New York, USA
• LIP OFECT AMINE ® commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL
• TRANSFECTAM® commercially available cationic liposomes comprising DOGS from Promega Corp., Madison, Wisconsin, USA.
• noncationic lipids used in the present invention can be any of a variety of neutral uncharged, zwitterionic or anionic lipids capable of producing a stable complex. They are preferably neutral, although they can alternatively be positively or negatively charged.
• noncationic lipids useful in the present invention include: phospholipid-related materials, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine (DOPC), dipalmitoyl-phosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-
• Additional nonphosphorous containing lipids are, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide and the like, diacylphosphatidylcholine, diacylphosphatidylethanolamme, ceramide, sphingomyelin, cephalin, and cerebrosides.
• stearylamine dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, is
• Noncationic lipids such as lysophosphatidylcholine and lysophosphatidylethanolamine may be present.
• Noncationic lipids also include polyethylene glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycol conjugated to phospholipids or to ceramides (referred to as PEG-Cer), as described in co-pending USSN 08/316,429, incorporated herein by reference.
• the noncationic lipids are diacylphosphatidylcholine (e.g., dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine and dilinoleoylphosphatidylcholine), diacylphosphatidylethanolamme (e.g., dioleoylphosphatidylethanolamine and pa mi oy o eoy p osp a i y e ano amme , cerami e or sp ingomye in.
• acy groups m these lipids are preferably acyl groups derived from fatty acids having Cio-C 24 carbon chains.
• the acyl groups are lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.
• the noncationic lipid will be 1,2-sn- dioleoylphosphatidylethanolamine, or egg sphingomyelin (ESM).
• the SPLP further comprises a bilayer stabilizing component (BSC).
• BSCs include, but are not limited to, polyamide oligomers, peptides, proteins, detergents, lipid-derivatives, PEG-lipids such as PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramides (see, U.S. Patent No. 5,885,613, which is incorporated herein by reference).
• the bilayer stabilizing component is a PEG-lipid, or an ATTA-lipid.
• the BSC is a conjugated lipid that inhibits aggregation of the SPLPs.
• Suitable conjugated lipids include, but are not limited to PEG-lipid conjugates, ATTA-lipid conjugates, cationic- polymer-lipid conjugates (CPLs) or mixtures thereof.
• the SPLPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate together with a CPL.
• the CPLs used in the present invention have the following architectural features: (1) a lipid anchor, such as a hydrophobic lipid, for incorporating the CPLs into the lipid bilayer; (2) a hydrophilic spacer, such as a polyethylene glycol, for linking the lipid anchor to a cationic head group; and (3) a polycationic moiety, such as a naturally occurring amino acid, to produce a protonizable cationic head group.
• a lipid anchor such as a hydrophobic lipid
• a hydrophilic spacer such as a polyethylene glycol
• a polycationic moiety such as a naturally occurring amino acid
• A is a lipid moiety such as an amphipathic lipid, a neutral lipid or a hydrophobic lipid that acts as a lipid anchor.
• Suitable lipid examples include vesicle-forming lipids or vesicle adopting lipids and include, but are not limited to, diacylglycerolyls, dialkylglycerolyls, N-N-dialkylaminos, l,2-diacyloxy-3-aminopropanes and l,2-dialkyl-3-aminopropanes.
• W is a polymer or an oligomer, such as a hydrophilic polymer or oligomer.
• the hydrophilic polymer is a biocompatable polymer that is nonimmunogenic or possesses low inherent immunogenicity.
• the hydrophilic polymer can be wea y an igenic i u wi appropria e a juvan s.
• ui a e nommmunogenic po ymers include, but are not limited to, PEG, polyamides, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers and combinations thereof.
• the polymer has a molecular weight of about 250 to about 7000 daltons. "Y" is a polycationic moiety.
• polycationic moiety refers to a compound, derivative, or functional group having a positive charge, preferably at least 2 positive charges at a selected pH, preferably physiological pH.
• Suitable polycationic moieties include basic amino acids and their derivatives such as arginine, asparagine, glutamine, lysine and histidine; spermine; spermidine; cationic dendrimers; polyamines; polyamine sugars; and amino polysaccharides.
• the polycationic moieties can be linear, such as linear tetralysine, branched or dendrimeric in structure.
• Polycationic moieties have between about 2 to about 15 positive charges, preferably between about 2 to about 12 positive charges, and more preferably between about 2 to about 8 positive charges at selected pH values.
• the selection of which polycationic moiety to employ may be determined by the type of liposome application which is desired.
• the charges on the polycationic moieties can be either distributed around the entire liposome moiety, or alternatively, they can be a discrete concentration of charge density in one particular area of the liposome moiety e.g., a charge spike. If the charge density is distributed on the liposome, the charge density can be equally distributed or unequally distributed. All variations of charge distribution of the polycationic moiety are encompassed by the present invention.
• the lipid "A,” and the nonimmunogenic polymer “W,” can be attached by various methods and preferably, by covalent attachment. Methods known to those of skill in the art can be used for the covalent attachment of "A” and “W.” Suitable linkages include, but are not limited to, amide, amine, carboxyl, carbonate, carbamate, ester and hydrazone linkages. It will be apparent to those skilled in the art that "A” and “W” must have complementary functional groups to effectuate the linkage. The reaction of these two groups, one on the lipid and the other on the polymer, will provide the desired linkage.
• the lipid is a diacylglycerol and the terminal hydroxyl is activated, for instance with NHS and DCC, to form an active ester, and is then reacted with a polymer which contains an amino group, such as with a polyamide (see, U.S. patent Application No. 09/218,988, filed December 22, 1998), an amide bond will form between the two groups.
• a polymer which contains an amino group such as with a polyamide
• "W” is bound, preferably covalently bound, to "Y".
• a covalent attachment of "W” to "Y” can be generated by comp emen ary reac ivi y o unc iona groups, one on e po ymer an e o er on e polycationic moiety.
• an amine functional group on "W” can be reacted with an activated carboxyl group, such as an acyl chloride or NHS ester, to form an amide.
• an activated carboxyl group such as an acyl chloride or NHS ester
• activated carboxyl groups include, but are not limited to, a carboxylic acid, a carboxylate ester, a carboxylic acid halide and other activated forms of carboxylic acids, such as a reactive anhydride.
• Reactive acid halides include for example, acid chlorides, acid bromides, and acid fluorides.
• the polycationic moiety can have a ligand attached, such as a targeting ligand or a chelating moiety for complexing calcium.
• a ligand attached such as a targeting ligand or a chelating moiety for complexing calcium.
• the cationic moiety maintains a positive charge.
• the ligand that is attached has a positive charge.
• Suitable ligands include, but are not limited to, a compound or device with a reactive functional group and include lipids, amphipathic lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immune stimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immiinoglobulins, functional groups, other targeting moieties, or toxins.
• Suitable chelating moieties for chelating or complexing the endosomal membrane destabilizer are described below. In certain preferred embodiments, other moieties are incorporated into the compounds of Formula I to form the compounds of Formula II:
• A is a lipid moiety such as, an amphipathic lipid, a neutral lipid or a hydrophobic lipid moiety.
• Suitable lipid examples include, but are not limited to, diacylglycerolyl, dialkylglycerolyl, N-N-dialkylamino, l,2-diacyloxy-3-aminopropane and 1 ,2-dialkyl-3 -aminopropane.
• X is a single bond or a functional group that covalently attaches the lipid to at least one ethylene oxide unit.
• Suitable functional groups include, but are not limited to, phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, carbonate, amido, thioamido, oxygen, NR wherein R is a hydrogen or alkyl group and sulfur.
• the number of ethylene oxide units can range from about 1 to about 160 and preferably from about 6 to about 50.
• Z is a single bound or a functional group that covalently attaches the ethylene oxide unit to the polycationic moiety.
• Suitable functional groups include, but are not limited to, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, amido, thioamido, NR wherem R is a member selected from the group consisting of hydrogen atom or alkyl group.
• the terminal ethylene oxide unit is directly attached to the polycationic moiety.
• "Y” is a polycationic moiety as described above in connection with Formula I.
• the index "n” is an integer ranging in value from about 6 to about 160.
• the residue is then purified by repeated precipitation of the chloroform mixture solution with diethyl ether until disappearance of the lipid using chromatography.
• the purified CPL conjugate is dissolved in a solvent, followed by addition of TFA, and the solution is stirred at room temperature. The solution can again be concentrated under a nitrogen stream.
• the residue is then purified by repeated precipitation of the mixture with diethyl ether to offer a lipid-PEG-NH 2 , such as a DSPE-PEG-NH 2 or, alternatively, DSPE-CPL- 1 with one protonizable cationic head group.
• the ratio of the phosphoryl-lipid anchor and the distal primary amine can then be measured by phosphate and fiourescamine assays as described herein.
• the number of protonizable amino groups can be increased to create a polycationic moiety.
• the polycationic moiety can be increase from about 2 to about 16 positive charges.
• the positive charges can be incorporated using any number of suitable polycationic moieties such as lysine, arginine, asparagine, glutamine, histidine, polyamines and derivatives or combinations thereof.
• the number of cationic groups, such as amino groups can be readily controlled during the CPL synthesis.
• the endosomal membrane destabilizer can be incorporated into the nucleic acid-lipid particle.
• the endosomal membrane destabilizer can be loaded into the nucleic-acid lipid particle using any of a number of different loading techniques (see, Examples I and II). Exemplar loading methods are disclosed, for example, in U.S. Patent No. 4,885,172, U.S. Patent No. 5,059,421, and U.S. Patent No. 5,171,578, the teachings of which are incorporated herein by reference.
• a particularly preferred ionophore-mediated loading process is disclosed and claimed in U.S. Patent No. 5,837,282, the teachings of which are incorporated herein by reference.
• a chelating moiety suitable for chelating the endosomal membrane destabilizer can be attached, linked or coupled to any of the lipid components of the SPLP, such as the CPL.
• the chelating moiety is a metal chelator.
• Metal chelators such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), ethylenebis(oxyethylenenitrilo)- tetraacetic acid (EGTA), l,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid, trans- l,2-cyclohexylenediamine-N,N,N',N' -tetraacetic acid, N 6 -carboxymethyl- N 3 , N 9 -[2,3- dihydroxy-N-methylpropylcarbamoylmethyl]-3,6,9-triaza ⁇ mdecanedioic acid, N - carboxymethyl- N , N -bis (methylcarbamoylmethyl)-3,6,9-triazaundecanedioic acid, N , N 6 -bis (carboxymethyl)- N 9 -3 -oxap
• nucleic Acid Component i e e mven ion is escri e in e examp es wi re erence o e use o plasmids, one of skill in the art will understand that the methods described herein are equally applicable to other larger nucleic acids or oligonucleotides.
• suitable nucleic acids include, but are not limited to, plasmids, antisense oligonucleotides, ribozymes as well as other poly- and oligo-nucleotides.
• nucleic acids which are useful in the present invention are typically nucleotide polymers having from 10 to 100,000 nucleotide residues.
• the nucleic acids are to be administered to a subject for the purpose of repairing or enhancing the expression of a cellular protein.
• the nucleic acid can carry a label (e.g., radioactive label, fluorescent label or colorimetric label) for the purpose of providing clinical diagnosis relating to the presence or absence of complementary nucleic acids.
• nucleic acids can be polymers of nucleic acids including genomic DNA, cDNA, mRNA or oligonucleotides containing nucleic acid analogs, for example, the antisense derivatives described in a review by Stein, et al, Science 261:1004-1011 (1993) and in U.S. Patent Nos. 5,264,423 and
• nucleic acids may encode transcriptional and translational regulatory sequences including promoter sequences and enhancer sequences.
• the nucleotide polymers can be single-stranded DNA or RNA, or double- stranded DNA or DNA-RNA hybrids.
• double-stranded DNA include structural genes, genes including 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.
• some single-stranded nucleic acids will preferably have some or all of the nucleotide linkages substituted with stable, nonphosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phosphoroselenate, or O-alkyl phosphotriester linkages.
• 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.
• sugar modifications include replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, azido groups or functionalized as ethers or esters.
• the entire sugar may be replaced with sterically and electronically similar structures, including aza-sugars and carbocyclic . sugar ana ogs.
• o i ica ions m e purine or pyrimi ine ase moie y inc u e, or examp e, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those of skill in the art.
• sequences for different proteins may be located on one strand or plasmid.
• Nonencoding sequences may be also be present, to the extent that 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. Patent No. 4,401,796; Caruthers, et al, U.S. Patent Nos.
• nucleic acids in the particles specificlly encompass vectors that are capable of being expressed in a host cell.
• 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.
• the expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid of interest to a promoter (which is either constitutive or inducible), incorporating the construct into an expression vector, and introducing the vector into a suitable host cell.
• Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters use u or regu a ion o e expression o e par icu ar nuc eic aci .
• e vec ors op ona y comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
• Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman and Smith (1979), Gene, 8: 81-97; Roberts et al.
• Vectors to which foreign nucleic acids are operably linked may be used to introduce these nucleic acids into host cells and mediate their replication and/or expression.
• “Cloning vectors” are useful for replicating and amplifying the foreign nucleic acids and obtaining clones of specific foreign nucleic acid-containing vectors.
• “Expression vectors” mediate the expression of the foreign nucleic acid.
• the particular vector used to transport a foreign gene into the cell is not particularly critical. Any of the conventional vectors used for expression in the chosen host cell may be used.
• An expression vector typically comprises a eukaryotic transcription unit or "expression cassette" that contains all the elements required for the expression of exogenous genes in eukaryotic cells.
• a typical expression cassette contains a promoter operably linked to the DNA sequence encoding a desired protein and signals required for efficient polyadenylation of the transcript.
• Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
• the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
• the other upstream promoter elements determine the rate at which transcription is initiated.
• Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus, the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic
• the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
• the termination region may be obtained from the same source as the promoter sequence or may be obtained from a different source.
• polyadenylation sequences are also commonly added to the vector construct.
• Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
• Termination and polyadenylation signals that are suitable for the present invention include those derived from S V40, or a partial genomic copy of a gene already resident on the expression vector.
• the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the transduced D ⁇ A.
• a number of animal viruses contain D ⁇ A sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate ac ors are provi e y genes ei er carrie on e p asmi or wi e genome o e os cell.
• the expression vectors of the present invention will typically contain both prokaryotic sequences that facilitate the cloning of the vector in bacteria as well as one or more eukaryotic transcription units that are expressed only in eukaryotic cells, such as mammalian cells.
• the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells.
• Selected genes are normally be expressed when the DNA sequence is functionally inserted into a vector. "Functionally inserted” means that it is inserted in proper reading frame and orientation and operably linked to proper regulatory elements. Typically, a gene will be inserted downstream from a promoter and will be followed by a stop codon, although production as a hybrid protein followed by cleavage may be used, if desired.
• SV40 vectors include pSVT7 and pMT2.
• Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
• exemplary vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculo virus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
• viral vectors such as retroviral vectors are useful for modifying eukaryotic cells because of the high efficiency with which the retroviral vectors transfect target cells and integrate into the target cell genome. Additionally, the retroviruses harboring the retroviral vector are capable of infecting cells from a wide variety of tissues.
• cells may be lipofected with adeno-associated viral vectors.
• adeno-associated viral vectors See, e.g., Methods in Enzymology, Vol. 185, Academic Press, Inc., San Diego, CA (DN. Goeddel, ed.) (1990) or M. Krieger (1990), Gene Transfer and Expression — A Laboratory Manual, Stockton Press, New York, NY, and the references cited therein.
• Adeno associated viruses require helper viruses such as adenovirus or herpes virus to achieve productive infection. In the absence of helper virus functions, AAV integrates (site-specifically) into a host cell's genome, but the integrated AAV genome has no pathogenic effect.
• the integration step allows the AAV genome to remain genetically intact un i e os s expose o e appropr a e env ronmen a con t ons e.g., a y ic e per virus), whereupon it re-enters the lytic life-cycle.
• Samulski (1993), Current Opinion in Genetic and Development, 3: 74-80, and the references cited therein provides an overview of the AAV life cycle. See also West etal. (1987), Virology, 160: 38-47; Carter et al. (1989), U.S. Patent No. 4,797,368; Carter et al.
• Plasmids designed for producing recombinant vaccinia such as pGS62, (Langford, C. L. et al. (1986), Mol. Cell. Biol, 6:3191-3199) may also be used.
• This plasmid consists of a cloning site for insertion of foreign nucleic acids, the P7.5 promoter of vaccinia to direct synthesis of the inserted nucleic acid, and the vaccinia TK gene flanking both ends of the foreign nucleic acid.
• the vector is genetically engineered to contain, in expressible form, a gene of interest that encodes a gene product of interest.
• Suitable classes of gene products include, but are not limited to, cytotoxic/suicide genes, immunomodulators, cell receptor ligands, tumor suppressors, and anti-angiogenic genes. The particular gene selected will depend on the intended purpose or treatment. Examples of such genes of interest are described below and throughout the specification. Cytotoxic/suicide genes are those genes that are capable of killing cells, causing apoptosis, or arresting cells in the cell cycle.
• genes include, but are not limited to, genes for immunotoxins, thymidine kinase, a cytochrome P450 2B1, a deoxycytidine kinase, or a cytosine deaminase.
• Agents such as acyclovir and ganciclovir (for thymidine kinase), cyclophosphoamide (for cytochrome P450 2B1), 5-fluorocytosine (for cytosine deaminase), are typically administered systemically in conjunction (e.g., simulatenously or nonsimulatenously) with the lipid-nucleic compositions of the present invention to achieve the desired cytotoxic or cytostatic effect.
• Immunomodulator genes are genes that modulate one or more immune responses.
• immunomodulator genes include cytokines such as growth factors (e.g., TGF- ⁇ ., TGF- ⁇ , EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-12, IL- 15, IL-20, etc.), interferons (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , etc.) and TNF.
• growth factors e.g., TGF- ⁇ ., TGF- ⁇ , EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.
• interleukins e.g., IL-2, IL-12, IL- 15, IL-20, etc.
• interferons e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , etc.
• Cell receptor ligands include ligands that are able to bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G-protein coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc), to modulate (e.g. inhibit, activate, etc.) the physiological pathway that the receptor is involved in (e.g., glucose level modulation, blood cell development, mitogenesis, etc.).
• cell receptor ligands include cytokines, .
• Tumor suppressor genes are genes that are able to inhibit the growth of a cell, particularly tumor cells. Thus, delivery of these genes to tumor cells is useful in the treatment of cancers. Tumor suppressor genes include, but are not limited to, p53 (Lamb et al, Mol. Cell. Biol. 6:1379-1385 (1986), Ewen et al, Science 255:85-87
• Anti-angiogenic genes are able to inhibit angiogenesis. These genes are particularly useful for treating those cancers in which angiogenesis plays a role in the pathological development of the disease. Examples of anti- angiogenic genes include, but are not limited to, endostatin (see e.g., U.S. Patent No.
• the vectors further usually comprise selectable markers which result in nucleic acid amplification such as the sodium, potassium ATPase, thymidine kinase, aminoglycoside phosphotransferase, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD (carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase), adenosine deaminase, dihydrofolate reductase, and asparagine synthetase and ouabain selection.
• high yield expression systems not involving nucleic acid amplification are also suitable, such as using a baculovirus vector in insect cells, with the encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
• nucleic acids other than plasmids 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 al, Science 261:1004-1011 (1993) and in U.S. Patent Nos. 5,264,423 and 5,276,019, the disclosures of which are incorporated herein by reference.
• the lipid-nucleic acid complexes of the prtesent invention may be used to transfer large (e.g., 50-5,000 kilobase) exogenous nucleic acids into cells.
• This aspect of lipofection is particularly advantageous since many genes which may be targets for gene therapy span over 100 kilobases (e.g., amyloid precursor protein (APP) gene, un ing ons c orea gene an arge omo ogous arge ing cons ruc s or ransgenes may e required for therapy.
• APP amyloid precursor protein
• Cells can be lipofected with an exogenous nucleic acid at high efficiency and with cell type specificity by contacting the cells with a receptor-recognition transfection complex comprising: (1) an exogenous nucleic acid, (2) a receptor-ligand protein ("rip") which is covalently linked to a poly cation, and (3) a cationic or neutral lipid.
• a receptor-recognition transfection complex comprising: (1) an exogenous nucleic acid, (2) a receptor-ligand protein ("rip") which is covalently linked to a poly cation, and (3) a cationic or neutral lipid.
• a combination of a polycation-linked receptor-recognition protein and a suitable cationic (or neutral) lipid can be used to transfect nucleic acids, and that the combination retains cell type targeting specificity conferred by the receptor-recognition protein and also exhibits high efficiency transfection conferred, in part, by the inclusion of a cationic lipid, neutral lipid, or lipopolyamine.
• the exogenous nucleic acid is typically dsDNA, ssDNA, ssRNA, dsRNA; most typically the exogenous nucleic acid is dsDNA such as a cloned DNA sequence in a cloning vector such as a plasmid or viral genome.
• dsDNA such as a cloned DNA sequence in a cloning vector such as a plasmid or viral genome.
• Multiple species of exogenous nucleic acid may be combined in a transfection complex, such as for co-transfection of unlinked nucleic acid sequences or to accomplish in vivo homologous recombination shuffling.
• the exogenous nucleic acid(s) are not capable of autonomous replication in cells which incorporate the transfection complex, and are either transiently expressed or are stably integrated into a host cell chromosome by homologous recombination or nonhomologous integration.
• at least one selectable marker e.g. , a neo R expression cassette
• an exogenous nucleic acid comprises a structural gene encoding a polypeptide to be expressed in a target cell which has incorporated the exogenous nucleic acid, and the structural gene usually is operably linked to appropriate cis-acting regulatory elements (e.g., promoter, enliancer, polyadenylation site).
• a typical receptor-recognition lipofection complex comprises a nucleic acid which comprises at least one transcriptional unit.
• the lipid nucleic acid particles of the invention can be designed to contain, in addition to the species of nucleic acid, a receptor-recognition molecule (rim), such as a protein.
• rim can be covalently bound to lipids that comprise the nucleic acid-lipid particle. Its presence on the particle increases the efficiency aand specificity with the particle contacts and enters target cells.
• a suitable rim is a nonimmunoglobulin protein that binds to a cell surface receptor of a target cell which mediates internalization of a transfection complex comprising the rlm-polycation conjugate by, for example, the process of endocytosis and/or membrane fusion.
• rim species typically are naturally- occurring physiological ligands which comprise a polypeptide portion (e.g., adhesion molecules such as ICAM-1, ICAM-2, ELAM-1, VCAM-1).
• a polypeptide portion e.g., adhesion molecules such as ICAM-1, ICAM-2, ELAM-1, VCAM-1).
• Viral proteins e.g., spike glycoproteins
• rim species for forming rlm-polycation conjugates.
• Examples also include viral glycoproteins which attach to cell surface receptors and lead to internalization and/or membrane fusion include the gB, gC, gD, gE, gH, and gl virion glycoproteins of HSV-1, and gpl20 of HIV-1.
• Fragments and analogs of naturally-occurring proteins may be used as well as full-length mature proteins as rim species in forming transfection complexes of the invention.
• fragments, analogs, and fusion proteins comprising a portion of an adhesion molecule or virion attachment protein which mediates attachment to a target cell may be used as rim species without other portions of the naturally-occurring full-length protein that are not essential for cell attachment and/or membrane fusion.
• a cytoplasmic tail peptide portion of a virion glycoprotein usually may be omitted and the resultant protein may still serve as a suitable rim.
• asialoglycoproteins galactose-terminal
• examples of asialoglycoproteins include asialoorosomucoid, asialofetuin, and desialylated vesicular stomatitis virus virion proteins. These can be formed by chemical or enzymatic desialylation of those glycoproteins that possess terminal sialic acid and penultimate galactose residues.
• rim species suitable for forming lipofection complexes that selectively target hepatocytes may be created by coupling lactose or other galactose-terminal carbohydrates (e.g., arabinogalactan) to nongalactose-bearing proteins by reductive lactosamination.
• lactose or other galactose-terminal carbohydrates e.g., arabinogalactan
• Other useful galactose-terminal carbohydrates for hepatocyte targeting include carbohydrate trees obtained from natural glycoproteins, especially tri- and tetra-antennary structures that contain either terminal galactose residues or that can be enzymatically treated to expose terminal galactose residues.
• rim species comprising mannose or mannose-6-phosphate, or complex carbohydrates comprising these terminal carbohydrate structures may be used. Since a variety of different cell surface receptors exist on the surfaces of mammalian cells, cell-specific targeting of nucleic acids to nonhepatic cells can involve lipofection complexes that comprise various rim species. For example, transferrin can be used as a suitable rim for forming receptor-recognition transfection complexes to cells expressing rans errin recep ors.
• er recep or igan s suc as po ypep i e ormones may be used to localize receptor-recognition transfection complexes to cells expressing the cognate receptor.
• the nucleic acid-lipid particles may comprise multiple rim species.
• an agent having membrane fusion activity e.g. , influenza virus hemagglutinin, HSV-1 gB and gD
• transfection methods generally comprise the steps of: (1) forming a nucleic acid-lipid-rlm particle consisting essentially of an exogenous nucleic acid, a polycation conjugate consisting essentially of a polycation linked to a nonimmunoglobulin receptor-recognition molecule that binds to a predetermined cell surface receptor, and a lipid component consisting essentially of a neutral or cationic lipid (optionally including a quaternary ammonium detergent and/or a lipopolyamine), and (2) contacting cells expressing the predetermined cell surface receptor with a composition comprising the receptor- recognition transfection complex under physiological transfection conditions which permit uptake of the exogenous nucleic acid into said cells.
• the rim is attached to the polycation by covalent linkage, frequently by covalent linkage through a crosslinking agent or by peptide linkage.
• the present invention provides lipid-nucleic acid particles produced via hydrophobic nucleic acid-lipid intermediate complexes.
• the complexes are preferably charge-neutralized. Manipulation of these complexes in either detergent-based or organic solvent-based systems can lead to particle formation in which the nucleic acid is protected.
• the present invention provides a method of preparing serum-stable plasmid- lipid particles in which the plasmid or other nucleic acid is encapsulated in a lipid bilayer and is protected from degradation. Additionally, the particles formed in the present invention are preferably neutral or negatively-charged at physiological pH. For in vivo applications, neutral particles are advantageous, while for in vitro applications the particles are more preferably negatively charged. This provides the further advantage of reduced aggregation over the positively-charged liposome formulations in which a nucleic acid can be encapsulated in cationic lipids.
• the particles made by the methods of this invention 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.
• a plasmid or other nucleic acid is contacted with a detergent solution of cationic lipids to form a coated plasmid complex.
• These coated plasmids can aggregate and precipitate.
• the presence of a detergent reduces this aggregation and allows the coated plasmids to react with excess lipids (typically, noncationic lipids) to form particles in which the plasmid or other nucleic acid is encapsulated in a lipid bilayer.
• excess lipids typically, noncationic lipids
• the particles are formed using detergent dialysis.
• 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 noncationic lipids with the coated plasmid-lipid complex to form a detergent solution comprising a plasmid-lipid complex and noncationic 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.
• 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.
• 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- ⁇ -D-glucopyranoside; and heptylthioglucopyranoside; with octyl ⁇ -D- glucopyranoside and Tween
• 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 ⁇ g/mL to about 1 mg/mL, preferably from about 25 ⁇ g/mL to about 200 ⁇ g/mL, and more preferably from about 50 ⁇ g/mL to about 100 ⁇ g/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.
• the plasmids and cationic lipids can be combined in the detergent solution and warmed to temperatures of up to about 37°C.
• the coated complexes can be formed at lower temperatures, typically down to about 4°C.
• the nucleic acid to lipid ratios (mass/mass ratios) in a formed SPLP will range from about 0.01 to about 0.08.
• the ratio of the starting materials also falls within this range because the purification step typically removes the unencapsulated nucleic acid as well as the empty liposomes.
• the SPLP preparation uses about 400 ⁇ g nucleic acid per 10 mg total lipid or a nucleic acid to lipid ratio of about 0.01 to about 0.08 and, more prederably, about 0.04, which corresponds to 1.25 mg of total lipid per 50 ⁇ g of nucleic acid.
• the detergent solution of the coated plasmid-lipid complexes is then contacted with noncationic lipids to provide a detergent solution of plasmid-lipid complexes and noncationic lipids.
• the noncationic lipids which are useful in this step include, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cardiolipin, and cerebrosides.
• the noncationic lipids are diacylphosphatidylcholine, diacylphosphatidylethanolamme, ceramide or sphingomyelin.
• the acyl groups in these lipids are preferably acyl groups derived from fatty acids having Cio-C 24 carbon chains. More preferably the acyl groups are lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.
• the noncationic lipid will be 1,2- sw-dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC) or egg phosphatidylcholine (EPC).
• DOPE 1,2- sw-dioleoylphosphatidylethanolamine
• POPC palmitoyl oleoyl phosphatidylcholine
• EPC egg phosphatidylcholine
• the plasmid-lipid particles will be fusogenic particles with enhanced properties in vivo and the noncationic lipid will be DOPE.
• the noncationic 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 USSN 08/316,429, incorporated herein by reference.
• the amount of noncationic lipid which is used in the present methods is typically about 2 to about 20 mg of total lipids to 50 ⁇ g of plasmid.
• the amount of total lipid is from about 5 to about 10 mg per 50 ⁇ g of plasmid.
• 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.
• 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.
• 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.
• the present invention provides a method for the preparation of serum-stable plasmid-lipid particles, comprising; a prepar g a mix ure comprising ca ionic ipi s an nonca ionic ipi s m an organic solvent;
• step (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.
• plasmids or nucleic acids
• cationic lipids or noncationic lipids
• noncationic lipids which are useful in this group of embodiments are as described for the detergent dialysis methods above.
• 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.
• plasmid Contacting the plasmid with the organic solution of cationic and noncationic 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.
• a first solution of plasmid which is typically an aqueous solution
• 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.
• the organic solvent is removed, thus fo ⁇ ning 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.
• the methods will further comprise adding nonlipid polycations which are useful to effect the transformation of cells using the present compositions.
• suitable nonlipid polycations include, hexadimethrine bromide . . . . so un er e ran name , om ric emica o., i wau ee, Wisconsin, 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.
• 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.
• the conjugating group i.e. phosphatidic acid or phosphatidylethanolamine
• an appropriately functionalized polyoxyethylene derivative i.e. phosphatidic acid or phosphatidylethanolamine
• phosphatidylethanolamine 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 formation of the lipid-nucleic acid complexes can be carried out either in a monophase system (e.g., a Bligh and Dyer monophase or similar mixture of aqueous and organic solvents) or in a two phase system with suitable mixing.
• a monophase system e.g., a Bligh and Dyer monophase or similar mixture of aqueous and organic solvents
• the cationic lipids and nucleic acids are each dissolved in a volume of the monophase mixture. Combination of the two solutions provides a single mixture in which the complexes form.
• the complexes can form in two-phase mixtures in which the cationic lipids bind to the nucleic acid (which is present in the aqueous phase), and "pull" it in to the organic phase.
• the present invention provides a method for the preparation of lipid-nucleic acid particles, comprising:
• nucleic acids contacting nucleic acids with a solution comprising noncationic lipids and a detergent to form a nucleic acid-lipid mixture;
• cationic lipids contacting cationic lipids with the nucleic acid-lipid mixture to neutralize a portion of the negative charge of the nucleic acids and form a charge-neutralized mixture of nucleic acids and lipids;
• the solution of noncationic lipids and detergent is an aqueous solution.
• Contacting the nucleic acids with the solution of noncationic lipids and detergent is typically accomplished by mixing together a first solution of nucleic acids and a second solution of the lipids and detergent.
• this mixing can take place by any number of methods, for example by mechanical means suc as y using vor ex mixers. re era y, e nuc eic aci so u ion is a so a e ergen solution.
• the amount of noncationic lipid which is used in the present method is typically determined based on the amount of cationic lipid used, and is typically of from about 0.2 to 5 times the amount of cationic lipid, preferably about 0.5 to 2 times the amount of cationic lipid used.
• the nucleic acid-lipid mixture thus formed is contacted with cationic lipids to neutralize a portion of the negative charge which is associated with the nucleic acids (or other polyanionic materials) present.
• the amount of cationic lipids used will typically be sufficient to neutralize at least 50% of the negative charge of the nucleic acid.
• the negative charge will be at least 70%) neutralized, more preferably at least 90%> neutralized.
• Cationic lipids which are useful in the present invention include, for example, DODAC, DOTMA, DDAB, DOTAP, DC-Choi and DMRIE. These lipids and related analogs have been described in co-pending USSN 08/316,399; U.S. Patent Nos.
• 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, New York, USA); LIPOFECTAMINE® (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising DOGS in ethanol from Promega Corp., Madison, Wisconsin, USA).
• LIPOFECTIN® commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, New York, USA
• LIPOFECTAMINE® commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL
• TRANSFECTAM® commercially available cationic lipids comprising DOGS in ethanol from Promega Corp., Madison, Wisconsin, USA.
• Contacting the cationic lipids with the nucleic acid-lipid mixture can be accomplished by any of a number of techniques, preferably by mixing together a solution of the cationic lipid and a solution containing the nucleic acid-lipid mixture. Upon mixing the two solutions (or contacting in any other manner), a portion of the negative charge associated with the nucleic acid is neutralized. Nevertheless, the nucleic acid remains in an uncondensed state and acquires hydrophilic characteristics.
• the detergent (or combination of detergent and organic solvent) is removed, thus forming the lipid-nucleic acid particles.
• the methods used to remove the detergent will typically involve dialysis. When organic solvents are present, removal is typically accomplished by evaporation at reduced pressures or by blowing a stream of inert gas (e.g., nitrogen or argon) across the mixture. . . . . e par ic es us orme wi ypica y e size rom a ou nm o severa microns.
• the lipid- nucleic acid particles can be sonicated, filtered or subjected to other sizing techniques which are used in liposomal formulations and are known to those of skill in the art.
• the methods will further comprise adding nonlipid polycations which are useful to effect the lipofection of cells using the present compositions.
• suitable nonlipid polycations include, hexadimethrine bromide (sold under the brandname POLYBRENE ® , from Aldrich Chemical Co., Milwaukee, Wisconsin, USA) or other salts of hexadimethrine.
• suitable polycations include, for example, salts of poly- L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine and polyethyleneimine. Addition of these salts is preferably after the particles have been formed.
• the present invention provides methods for the preparation of lipid-nucleic acid particles, comprising:
• the solution of step (a) is a monophase. In another group of embodiments, the solution of step (a) is two-phase.
• the cationic lipids are DODAC, DDAB, DOTMA, DOSPA, DMRIE, DOGS or combinations thereof.
• the noncationic lipids are ESM, DOPE, polyethylene glycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modified phospholipids or PEG-modified ceramides) or combinations thereof.
• the organic solvents are methanol, chloroform, methylene chloride, ethanol, diethyl ether or combinations thereof.
• the nucleic acid is a plasmid;
• the cationic lipid is DODAC, DDAB, DOTMA, DOSPA, DMRIE, DOGS or combinations thereof;
• the noncationic lipid is ESM, DOPE, polyethylene glycol-based polymers or com ina ions ereo ; an e orgamc so ven s me ano , c oro orm, me y ene c ori e, ethanol, diethyl ether or combinations thereof.
• contacting the nucleic acids with the cationic lipids is typically accomplished by mixing together a first solution of nucleic acids and a second solution of the lipids, preferably by mechanical means such as by using vortex mixers.
• the resulting mixture contains complexes as described for one aspect of the invention above.
• These complexes are then converted to particles by the addition of noncationic lipids and the removal of the organic solvent.
• the addition of the noncationic lipids is typically accomplished by simply adding a solution of the noncationic lipids to the mixture containing the complexes. A reverse addition can also be used. Subsequent removal of organic solvents can be accomplished by methods known to those of skill in the art and also described above.
• the amount of noncationic lipids which is used in this aspect of the invention is typically an amount of from about 0.2 to 5 times the amount (on a mole basis) of cationic lipids which was used to provide the charge-neutralized lipid-nucleic acid complex.
• the amount is from 0.5 to 2 times the amount of cationic lipids used.
• the present invention provides lipid-nucleic acid particles which are prepared by the methods described above.
• the lipid-nucleic acid particles are either net charge neutral or carry an overall charge which provides the particles with greater gene lipofection activity.
• the nucleic acid component of the particles is a nucleic acid which encodes a desired protein or blocks the production of an undesired protein.
• the nucleic acid is a plasmid
• the noncationic lipid is egg sphingomyelin
• the cationic lipid is DODAC.
• SPLP-CPLs A variety of general methods for making SPLP-CPLs (CPL-containing SPLPs) are discussed herein.
• Two general techniques include "post-insertion” technique, that is, insertion of a CPL into for example, a pre-formed SPLP, and the "standard” technique, wherein the CPL is included in the lipid mixture during for example, the SPLP formation steps.
• the post-insertion technique results in SPLPs having CPLs mainly in the external face of the SPLP bilayer membrane, whereas standard techniques provide SPLPs having CPLs on both internal and external faces.
• post-insertion involves forming SPLPs (by any method), and incubating the pre-formed SPLPs in the presence of CPL under appropriate conditions (preferably 2-3 hours at 60°C).
• the CPL can be inserted into the external leaflet of the recipient vesicle, giving final concentrations up to about 5 to 10 mol % (relative o o a ipi . e me o is especia y use u or ves c es ma e om p osp o ip s w ic can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-Ceramide).
• PEG-lipids such as PEG-Ceramide
• the CPL-SPLPs of the present mvention can be formed by extrusion.
• all of the lipids including the CPL are co-dissolved in chloroform, which is then removed under nitrogen followed by high vacuum.
• the lipid mixture is hydrated in an appropriate buffer, and extruded through two polycarbonate filters with a pore size of 100 nm.
• the resulting SPLPs contain CPL on both of the internal and external faces.
• the formation of CPL- SPLPs can be accomplished using a detergent dialysis or ethanol dialysis method, for example, as discussed in U.S. Patent Nos. 5,976,567 and 5,981,501, both of which are incorporated herein by reference.
• the nucleic acid-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.
• a physiologically-acceptable carrier such as physiological saline or phosphate buffer
• 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.
• the pharmaceutical carrier is generally added following particle formation.
• the particle can be diluted into pharmaceutically acceptable carriers such as normal saline.
• 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.
• 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.
• particles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration.
• PEG-lipid conjugates such as PEG-ceramides or PEG-PE, ganglioside G M i-modified lipids or ATTA-lipids to the particles.
• Addition of such components prevents particle aggregation and provides a means or increasing circu a ion i e ime an increasing e e ivery o e ipi -nuc eic aci particles to the target tissues.
• the concentration of the component in the particle will be about 1-20 %> and, more preferably from about 3-10 %.
• compositions may be sterilized by conventional, well known sterilization techniques.
• Aqueous solutions can 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 can 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, and calcium chloride.
• 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.
• lipid-nucleic acid particles can be incorporated into a broad range of topical dosage forms including but not limited to gels, oils, emulsions and the like.
• the suspension containing the nucleic acid-lipid particles can be formulated and administered as topical creams, pastes, ointments, gels, lotions and the like.
• the present invention also provides lipid-nucleic acid particles in kit form.
• the kit will typically be comprised of a container which is compartmentalized for holding the various elements of the lipid-nucleic acid particles and the endosomal membrane destabilizer (e.g., calcium ions).
• the kit will contain the compositions of the present inventions, preferably in dehydrated form, with instructions for their rehydration and administration.
• 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.
• the serum-stable nucleic acid-lipid particles of the present invention are useful for the introduction of nucleic acids into cells. Accordingly, the present mvention also provides methods for introducing a nucleic acids (e.g., a plasmid) into a cell. The methods are carrie ou in vi ro or in vivo y irs orming e par ic es as escri e a ove, en contacting the particles with the cells for a period of time sufficient for transfection to occur.
• a nucleic acids e.g., a plasmid
• the nucleic acid-lipid particles of the present invention can be adsorbed to almost any cell type with which they are mixed or contacted. 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.
• 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.
• the cells will be animal cells, more preferably mammalian cells, and most preferably human cells.
• Contact between the cells and the lipid-nucleic acid particles, when carried out in vitro, takes place in a biologically compatible medium.
• concentration of particles varies widely depending on the particular application, but is generally between about 1 ⁇ mol and about 10 mmol.
• Treatment of the cells with the nucleic acid-lipid particles is generally carried out at physiological temperatures (about 37° C) for periods of time of from about 1 to 48 hours, preferably of from about 2 to 4 hours.
• a lipid-nucleic acid particle suspension is added to 60-80% confluent plated cells having a cell density of from about 10 to about 10 5 cells/mL, more preferably about 2 x 10 4 cells/mL.
• the concentration of the suspension added to the cells is preferably of from about 0.01 to 0.2 ⁇ g/mL, more preferably about 0.1 ⁇ g/mL.
• compositions of the present invention can also be used for the in vivo gene transfer, using methods which are known to those of skill in the art.
• 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.
• CMV cytomegalovirus
• CAT chloramphenicol acetyltransferase
• CTR cystic fibrosis transmembrane conductance regulator
• 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.
• parenterally i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
• the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
• a bolus injection see Stadler, et al, U.S. Patent 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); Marmino, et al, Biotechniques 6:682-690 (1988); Nicolau, et al, Crit Rev. Ther. Drug Carrier Syst. 6:239-271 (1989), and Behr, Ace. Chem. Res. 26:274-278 (1993). Still other methods of administering lipid-based therapeutics are described in, for example, Raliman et al, U.S. Patent No. 3,993,754; Sears, U.S. Patent No.
• 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.
• 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.
• 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 metrazamide imaging of the spinal cord.
• the preparations may be administered through endoscopic devices.
• the lipid-nucleic acid 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, Mary Ann 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, nonhuman primates, dogs, cats, cattle, horses, sheep, and the like.
• the amount of particles administered will depend upon the the ratio of nucleic acid to lipid; the particular nucleic acid used, the disease state being diagnosed; the age, weight, and condition of the patient 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 or about 10 8 -10 10 particles per injection.
• Some methods of gene therapy serve to compensate for a defect in an endogenous gene by integrating a functional copy of the gene into the host chromosome.
• the inserted gene replicates with the host DNA and is expressed at a level to compensate for the defective gene.
• Diseases amendable to treatment by this approach are often characterized by recessive mutations. That is, both copies of an endogenous gene must be defective for symptoms to appear.
• Such diseases include cystic fibrosis, sickle cell anemia, ⁇ -thalassemia, phenylketonuria, galactosemia, Wilson's disease, hemochromatosis, severe combined immunodeficiency disease, alpha- 1-antitrypsin deficiency, albinism, alkaptonuria, lysosomal storage diseases, Ehlers-Danlos syndrome, hemophilia, glucose-6-phosphate dehydrogenase deficiency, agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, fragile X-syndrome, and the like.
• Other recessive mutations are known in the art, and the use of the methods of the present invention to treat them is contemplated herein.
• cells are removed from a patient suffering from the disease and contacted with a lipid- vector complex in vitro.
• Cells should be removed from a tissue type in which disease symptoms are manifested.
• the vector used includes a selective marker, cells having internalized and expressed the marker can be selected. Particularly if selection is not per ormed, it is important that the frequency of gene transfer into cells be high, for example, at least about 1, 5, 10, 25 or 50% of cells.
• the lipid- vector complex can be introduced directly into a patient as a pharmaceutical composition.
• the complex is delivered to the tissue(s) affected by the genetic disorder being treated in a therapeutically effective dose.
• a therapeutically effective dose is an amount sufficient to cure, or at least partially arrest, the symptoms of the disease and its complications.
• Effective doses of the compositions of the present invention, for the treatment of the above described conditions will vary depending upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages will need to be titrated to optimize safety and efficacy. Doses ranging from about 10 ng to 1 g, 100 ng to 100 mg, 1 ⁇ g to 10 mg, or 30-300 ⁇ g DNA per patient are typical.
• nucleic acid-lipid complexes can also be used to transfect embryonic stem cells or zygotes to achieve germline alterations.
• ' See Jaenisch, Science, 240:1468-1474 (1988); Gordon et al, Methods Enzymol. 101, 414 (1984); Hogan et al, Manipulation of the Mouse Embryo: A Laboratory Manual, C.S.H.L. NN. (1986); and Hammer et al, Nature 315:680 (1985); Gandolfi et al, J. Reprod. Fert. 81:23-28 (1987); Rexroad et al, J. Anim. Sci.
• cystic fibrosis is a usually fatal recessive genetic disease, having a high incidence in Caucasian populations.
• the gene responsible for this disease was isolated by Riordan et al, Science 245:1059-1065 (1989). It encodes a protein called the cystic fibrosis transmembrane conductance regulator (CFTR) which is involved in the transfer of chloride ions (Cf) through epithelial cell membranes. Mutations in the gene cause defects of CI " secretion in epithelial cells leading to the various clinical manifestations.
• CF cystic fibrosis transmembrane conductance regulator
• a vector containing a coding sequence for a functional CFTR gene product can be complexed with lipid, and optionally, a pharmaceutical excipient and introduced into the patient via nasal administration so that the vector-lipid composition reaches the lungs.
• the dose of vector- lipid complex is preferably about 10 8 -10 10 particles.
• defects in the ⁇ or ⁇ globin genes can be compensated for by ex vivo treatment of hemopoietic stem cells with an nucleic acid-lipid complex containing a functional copy of the gene.
• the gene integrates into the stem cells which are then reintroduced into the patient.
• Defects in the gene responsible for Fanconi Anemia Complement Group C can be treated by an analogous strategy (see Walsh et al, J. Clin. Invest. 94:1440-1448 (1994)).
• Li-Fraumeni syndrome is a hereditary condition in which individuals receive mutant p53 alleles, resulting in the early onset of various cancers (Harris, Science 262:1980-1981 (1993) Frecios et al, PNAS 89:6413-6417 (1992); Malkin et al, Science 250:1233 (1990)).
• a tumor suppressor gene in a cancerous cell or a cell at risk of becoming cancerous is effective to prevent, arrest and or reverse cellular proliferation and other manifestations of the cancerous state.
• Suitable tumor suppressor genes for use in the invention include p53 (Buchman et al, Gene 70:245-252 (1988)), APC, DCC, Rb, WTl, and NFl (Marx, Science 260:751-752 (1993); Marshall, Cell 64:313-326 (1991)).
• Lipid-nucleic acid complexes bearing a functional copy of a tumor suppressor gene are usually administered in vivo by the route most proximal to the intended site of action. For example, skin cancers can be treated by topical administration and leukemia by intravenous administration.
• Methods of gene therapy using the nucleic acid-lipid complexes of the invention can also be used for prophylactic or therapeutic treatment of patients or cells, infected with or at risk of being infected with, a pathogenic microorganism, such as HIV.
• a pathogenic microorganism such as HIV.
• T e e ectiveness o ant sense molecules in blocking target gene functions o impeding virus replication has been demonstrated in a number of different systems (Friedman et al, Nature 335:452-54 (1988), Malim et al, Cell 58:205-14 (1989) & Trono at al., Cell 59:113-20 (1989)).
• the vector used includes a DNA segment encoding an antisense transcript, which is complementary to a segment of the genome from the pathogenic microorganism.
• the segment should preferably play an essential role in the lifecycle of the microorganism, and should also be unique to the microorganism (or at least absent from the genome of the natural genome of a patient undergoing therapy).
• suitable sites for inhibition on the HIV virus includes TAR, REV or nef (Chatterjee et al, Science 258:1485-1488 (1992)).
• Rev is a regulatory RNA binding protein that facilitates the export of unspliced HIV pre mRNA from the nucleus. Malim et al, Nature 338:254 (1989). Tat is thought to be a transcriptional activator that functions by binding a recognition sequence in 5' flanking mRNA. Karn & Graeble, Trends Genet. 8:365 (1992).
• the nucleic acid-lipid complex is introduced into leukocytes or hemopoietic stem cells, either ex vivo or by intravenous injection in a therapeutically effective dose.
• the treatment can be administered prophylactically to HIV " persons, or to persons already infected with HIV.
• Analogous methods are used for suppressing expression of endogenous recipient cell genes encoding adhesion proteins. Suppression of adhesion protein expression in useful in aborting undesirable inflammatory responses.
• Adhesion proteins that can be suppressed by antisense segments present in seelcted vectors include integrins, selectins, and immunoglobulin (Ig) superfamily members (see Springer, Nature 346:425-433 (1990). Osborn, Cell 62:3 (1990); Hynes, Cell 69:11 (1992)).
• Integrins are heterodimeric transmembrane glycoproteins consisting of an ⁇ chain (120-180 kDa) and a ⁇ chain (90-110 kDa), generally having short cytoplasmic domains.
• LFA-1 The three known integrins, LFA-1, Mac- 1 and P150,95, have different alpha subunits, designated CDl la, CDl lb and CDl lc, and a common beta subunit designated CDl 8.
• LFA-1 ( ⁇ 2 ) is expressed on lymphocytes, granulocyte and monocytes, and binds predominantly to an Ig-family member counter- receptor termed ICAM-1 (and perhaps to a lesser extent ICAM-2).
• ICAM-1 is expressed on many cells, including leukocytes and endothelial cells, and is up-regulated on vascular endothelium by cytokines such as TNF and IL-1.
• Mac-1 ( ⁇ M ⁇ a) s distributed on neutrophils and monocytes, and also binds to ICAM-1 (and possibly ICAM-2).
• the selectins consist of L- selectin, E-selectin and P-selectin. 5.
• compositions and methods of the present invention are used to treat a wide variety of cell types, in vivo and in vitro.
• those most often targeted for gene therapy are hematopoietic precursor (stem) cells.
• Other cells include those of which a proportion of the targeted cells are nondividing or slow dividing. These include, for example, fibroblasts, keratinocytes, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, quiescent lymphocytes, terminally differentiated cells, slow or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, etc.
• the methods and compositions can be employed with cells of a wide variety of vertebrates, including mammals, and especially those of veterinary importance, e.g, canine, feline, equine, bovine, ovine, caprine, rodent, lagomorph, swine, etc., in addition to human cell populations.
• Gene therapy relies on the efficient delivery of therapeutic genes to target cells.
• Most of the somatic cells that have been targeted for gene therapy e.g., hematopoietic cells, skin fibroblasts and keratinocytes, hepatocytes, endothelial cells, muscle cells and lymphocytes, are normally nondividing.
• Retroviral vectors which are the most widely used vectors for gene therapy, unfortunately require cell division for effective transduction (Miller et al, Mol. Cell. Biol. 10:4239-4242 (1990)). This is also true with other gene therapy vectors such as the adeno-associated vectors (Russell et al, Proc. Natl. Acad. Sci.
• nucleic acid construct that encodes a gene of interest
• Nucleic acids and proteins are detected and quantified herein by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodif usion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
• the detection of nucleic acids proceeds by well known methods such as Southern analysis, northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and affinity chromatography.
• nucleic acid hybridization format is not critical.
• a variety of nucleic acid hybridization formats are known to those skilled in the art.
• common formats include sandwich assays and competition or displacement assays.
• Hybridization techniques are generally described in "Nucleic Acid Hybridization, A Practical Approach,” Ed. Hames, B.D. and Higgins, S.J., IRL Press, 1985.
• the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected.
• a nucleic acid amplification system which multiplies the target nucleic acid being detected.
• In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q ⁇ -replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987), U.S. Patent No.
• the select sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
• Oligonucleotides for use as probes e.g., in in vitro amplification methods, for use as gene probes, or as inhibitor components are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts., 22(20): 1859-1862 (1981), e.g., using an automated synthesizer, as described in Needham-VanDevanter et al, Nucleic Acids Res., 12:6159-6168 (1984).
• oligonucleotides Purification of oligonucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier, J Chrom., 255:137-149 (1983). The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology, 65:499-560.
• In situ hybridization assays are well known and are generally described in Angerer et al, Methods Enzymol, 152:649-660 (1987).
• in situ hybridization assay cells are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labelled.
• the probes are preferably labelled with radioisotopes or fluorescent reporters.
• the expression of the gene of interest to produce a product may be detected or quantified by a variety of methods. Preferred methods involve the use of specific antibodies.
• Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of at least about .1 mM, more usually at least about 1 ⁇ M, preferably at least about .1 ⁇ M or better, and most typically and preferably, .01 ⁇ M or better.
• the presence of a desired polypeptide (including peptide, transcript, or enzymatic digestion product) in a sample may be detected and quantified using Western blot analysis.
• the technique generally comprises separating sample products by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with labeling antibodies that specifically bind to the analyte protein.
• the labeling antibodies specifically bind to analyte on the solid support.
• These antibodies are directly labeled, or alternatively are subsequently detected using labeling agents such as antibodies (e.g., labeled sheep anti-mouse antibodies where the antibody to an analyte is a murine antibody) that specifically bind to the labeling antibody.
• N,N-dioleyl-N,N-dimethylammonium chloride was obtained from Dr. S. Ansell and l-O-(2-( ⁇ -methoxyethyleneglycol)succinoyl)-2-N- arachidoylsphingosine (PEG-CerC o) was synthesized by Dr. Z. Wang at Inex Pharmaceuticals Corporation (Burnaby, BC).
• l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) and l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were obtained from Northern Lipids (Vancouver, BC).
• l,2-dioleoyl-sn-glycero-3-(phospho-L- serine) (DOPS) and l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl) (Rh-DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol (Choi), octylglucopyranoside (OGP), HEPES, MgCl 2 , and NaCl were o aine om igma emica o. . ouis, .
• ep arose -o - anio c- exchange column and Sepharose CL-4B sizing column materials were obtained from Sigma Chemical Co. (St. Louis, MO).
• the luciferase assay kit was purchased from Promega Corp. (Madison, WI).
• Picogreen dsDNA detection reagent was obtained from Molecular Probes (Eugene, OR).
• Plasmid DNA (pCMVLuc) coding for the luciferase reporter gene under the control of the human CMV immediate early promoter-enhancer element was obtained from Inex Pharmaceuticals Corporation (Burnaby, BC).
• Bovine hamster kidney (BHK) cells were obtained from the American Tissue Culture Collection (ATCC CCL-10, Rockville, MD) and cultured in Dulbecco modified Eagle medium (DMEM) supplement with 10%> fetal bovine serum (FBS), 100 U/ml of penicillin and 100 ⁇ g/ml of streptomycin. BHK cells were maintained as a monolayer at 37°C in a humidified atmosphere containing 5.0% CO 2 .
• DMEM Dulbecco modified Eagle medium
• FBS fetal bovine serum
• streptomycin 100 U/ml bovine serum
• BHK cells were maintained as a monolayer at 37°C in a humidified atmosphere containing 5.0% CO 2 .
• SPLP Preparation of SPLP.
• SPLP were prepared as described by Wheeler, et al, Gene Therapy 6:271-281 (1999)) with some modifications. Briefly, a total of 10 ⁇ moles of DODAC, DOPE, PEG-CerC 20 (7:83:10; mol/mol/mol) were dissolved in chloroform and dried under a stream of nitrogen gas. Residual solvent was removed under high vacuum for 2 h. The resulting lipid film was hydrated in 1 ml of HBS buffer (20 mM HEPES and 150 mM NaCl, pH 7.5) containing 0.2 M OGP with continuous vortexing.
• HBS buffer 20 mM HEPES and 150 mM NaCl, pH 7.5
• Plasmid DNA 400 ⁇ g/ml was added to the hydrated lipids and the mixtures were dialysed against HBS buffer for 36 to 48 h with 2 buffer changes.
• Nonencapsulated plasmid was removed by DEAE anion exchange cliromatography and empty lipid vesicles were removed by employing a sucrose density gradient as previously described (Mok, et al. , Biochimica et Biophysica Acta 1419:137-150 (1999)).
• SPLP were initially prepared in HBS buffer containing 30 mM sodium citrate as described previously (Zhang, et al, Gene Therapy 6: 1438-1447 (1999)). SPLP were characterized with respect to plasmid entrapment using a previously described Picogreen assay (Zhang, et al, Gene Therapy 6:1438-1447 (1999)) and sized using quasielastic light scattering.
• Lipid uptake was determined by comparing lysate fluorescence to that of a lipid standard normalizing it to the total cellular protein. To determine the intracellular SPLP localization, fluorescence microscopy was employed. Cells were transfected with vesicles labeled with 4 mol% Rh-DOPE. The transfection media was replaced with complete media prior to analysis under the fluorescence microscope.
• 31 P NMR spectroscopy Solid-state 31 P NMR spectra were recorded with broad-band decoupling at 81.02 MHz on a Bruker MSL 200 spectrometer, using a 3.8- ⁇ s 60° pulse and a 1.5-s repeat time. The free induction decay (FID) was accumulated over 2500-3000 scans and was Fourier transformed with 50-Hz line broadening. Phospholipid mixtures (25 ⁇ mol of total phospholipid) were dispersed by vortex mixing in 2 ml of buffer (20 mM HEPES buffer, pH 7.4). Increasing concentrations of Ca 2+ were titrated into the vesicles by adding aliquots of 200 mM CaCl stock.
• pelleted cells were lysed by incubating with 250 ⁇ l of lysis buffer (10 mM Tris, pH 7.5, 0.5% SDS, 1 mM EDTA) containing Pronase E at 1 mg/ml (Sigma) overnight at 37°C.
• DNA genomic DNA and delivered plasmid DNA
• DNA recovery was determined by measuring the absorbance at 260 nm.
• SPLP (DODAC/DOPE/PEG- CerC20/Rd-DOPE, 10:79.5:10:0.5, mol/mol/mol) were initially prepared in citrate buffer (150 mM sodium citrate and 150 mM citric acid) at pH 4.
• citrate buffer 150 mM sodium citrate and 150 mM citric acid
• Nonencapsulated plasmid was removed by DEAE anion exchange chromatography equilibrated in HBS buffer (pH 7.5) and empty lipid vesicles were removed by employing a sucrose density gradient as previously described (Mok, et al, Biochimica et Biophysica Acta 1419:137-150 (1999)).
• Ca 2+ loading was performed by incubation of the DNA-loaded vesicles with 2.5 mM CaCl 2 and the ionophores A23187 (0.1 ⁇ g/ ⁇ mole lipids) for 30 min at room temperature. Unloaded Ca and ionophores were removed by dialysis in HBS buffer with 2 buffer changes. Internal Ca 2+ concentrations were determined in the absence and presence of TX-100 (0.2%) by employing the membrane nonpermeant absorbant indicator Asenazo III (0.1 mM in 10 mM HEPES u er, p , agains a a s an ar curves o nmo es . sor ances a nm were measured as an indicator of Ca 2+ presence.
• SPLP were characterized with respect to plasmid entrapment using a previously described Picogreen assay (Zhang, et al, Gene Therapy 6:1438-1447 (1999)) and sized using quasielastic light scattering. Internal concentrations of Ca 2+ were found to be ⁇ 175 mM.
• SPLP Prior to CPL insertion, SPLP were prepared as described in the previous section with some modification. SPLP containing total of 10 ⁇ moles of DODAC, DOPE, PEG-CerC 20 , and Rd-DOPE (7:82.5:10:0.5; mol/mol/mol/mol) were hydrated in 1 ml of HBS buffer (20 mM HEPES and 150 mM NaCl, pH 7.5) containing 0.2 M OGP with continuous vortexing. Plasmid DNA (400 ⁇ g/ml) was added to the hydrated lipids and the mixtures were dialysed against HBS buffer for 36 to 48 h with 2 buffer changes. Nonencapsulated plasmid was removed by DEAE anion exchange chromatography.
• CPL stocks in methanol labeled with a dansyl fluorescence marker were added to the SPLP to give the desired molar ratio (up to 4 mol%> CPL relative to vesicle lipid).
• CPL and SPLP were incubated for up to 3 h at 60°C, and cooled on ice to room temperature. Both empty lipid vesicles and noninserted CPL were removed by employing a sucrose density gradient. The insertion levels of CPL were quantitated by using the Perkin Elmer Luminescence Spectrophotometer.
• %-insertion ([D/R] f )*100/(D/R)i CPL-SPLP were further characterized with respect to plasmid entrapment using a previously described Picogreen assay (Zhang et al, 1999) and sized using quasielastic light scattering.
• SPLP are stable in the presence of Ca 2+ .
• SPLP with PEG-CerC 20 are highly stable systems that exhibit extended circulation times in vivo, protect encapsulated plasmid from external nucleases, and do not interact readily with cells (Wheeler, et al, Gene Therapy 6:271-281 (1999); Mok, et al, Biochimica et Biophysica Acta 1419:137-150 (1999); Monck, et al, Journal of Drug Targeting 7:439-452 (2000)). It was therefore important to
• Plasmid release was assayed over 24 h employing the Picogreen assay. No plasmid release was observed. . a oes no in uence e ce u ar up a e o - . ie a i i y o Ca 2+ to enhance the transfection activity of plasmid DNA-cationic lipid complexes has been attributed to an increase in the uptake of the complexes into cells in the presence of Ca 2+ (Lam, et al, Biochim BiophysActa 1463:279-290 (2000)).
• the low transfection potencies of SPLP as compared to complexes arise, at least in part, from very low levels of cellular uptake of SPLP (Mok, et ah, Biochimica et Biophysica Acta 1419:137- 150 (1999)). It was therefore of interest to determine whether Ca 2+ stimulated SPLP transfection potencies by increasing SPLP uptake into cells.
• SPLP containing 0.5 mol%> Rh- DOPE were employed to determine SPLP uptake into BHK cells in the presence of up to 12 mM Ca 2+ as described in the above Materials and Methods. The SPLP were incubated with cells for 4, 8, and 24 h and the levels of intracellular lipid determined.
• Lipid uptake at each time-point was normalized against total cell protein in order to account for cell growth. As shown in Figure 2, the results indicate that Ca 2+ did not significantly increase the cellular uptake of SPLP even though the transfection potencies of the SPLP varied by several hundred-fold over the range of Ca 2+ concentrations tested.
• Rh-labeled SPLP were incubated on BHK cells in the presence and absence of 10 mM Ca 2+ and the cell morphology was examined at 8 h by fluorescence microscopy. Similar levels of rhodamine fluorescence were detected in the absence or presence of Ca 2+ , in agreement with the quantitative measurements of SPLP uptake noted in the previous section. However, as shown in Figure 3, the appearance of the cells as detected by fluorescence microscopy was quite different in the presence or absence of Ca 2+ . Although some punctate structures are observed, BHK cells containing the fluorescently-labeled SPLP ex i i e a more use pa ern w en a was nc u e . n e a sence o a , t e fluorescence pattern was largely punctate, consistent with SPLP retention in the endosomal compartments.
• Ca 2+ can destabilize lipid bilayers containing acidic lipids such as phosphatidylserine (PS) in combination with unsaturated PEs by inducing the nonbilayer hexagonal H ⁇ phase structure (Hope, et al, FEBS Letters 107:323-326 (1979); Tilcock, et al, Biochimica et Biophysica Acta 641:189-201 (1981)). It has also been shown that Ca 2+ can induce Hn phase structure in related systems
• PC phosphatidylcholine
• addition of Ca to mixtures of DOPC/DOPE/DOPS/Cholesterol (1:1:1:3; molar ratios) also triggers bilayer to hexagonal H ⁇ phase transitions (Tilcock, et al, Biochemistry 23:2696-2703 (1984)). It is us possi e a a s imu a es rans ec ion y ac ing m concer wi e ca omc lipid in the SPLP to destabilize the lipid bilayer of endosomal membranes.
• SPLP Effect of Ca 2+ on improved SPLP systems.
• One limitation of SPLP is that the system is not optimally taken by cells as a result of limited cationic lipid and presence o on e vesic es o , et a ., ioc imica et iop ysica c a : - (1999)).
• One straightforward way to increase the positive charges is by increasing the cationic content (Zhang, et ah, Gene Therapy 6:1438-1447 (1999)).
• transfection efficiencies increased with SPLP increased DODAC concentrations, and such systems are thus compromised with lower DNA encapsulation.
• CPL cationic poly(ethylene glycol) lipid conjugates
• the mechanism whereby Ca 2+ stimulates the transfection potency of SPLP must account for several observations.
• the enhanced transfection appears to result from higher intracellular levels of intact plasmid in the presence of Ca 2+ ; these higher levels of plasmid do not appear to arise from increased uptake of SPLP into cells, however.
• the process is associated with a reduction in the "punctate" appearance of cells following uptake of fluorescently labeled SPLP.
• the effect is Ca 2+ specific.
• the first two observations are clearly consistent with enhanced endosomal destabilization of the BHK cells following endocytosis of SPLP. The question thus remaining is how Ca 2+ could promote this es a i iza ion in a speci ic manner.
• n is regar
• ere is presen y no consensus as o ow endosomes can be destabilized to enhance release of their contents, however a number of leading observations have been made. Chief amongst these is the observation that cationic lipids can dramatically enhance the intracellular delivery of macromolecules such as plasmids and antisense oligonucleotides (Bennett, et ah, Mol. Pharmacol. 41:1023-1033 (1992); Barron, et ah, Gene Ther.
• Ca 2+ can induce H ⁇ phase structure in previously bilayer lipid systems containing anionic lipids, and that this effect is Ca 2+ -specific, as other cations such as Mg 2+ either cannot induce H ⁇ structure or require higher concentrations to produce similar effects (Tilcock, et ah, Biochemistry, 23:2696-2703 (1984)).
• Ca 2+ can induce H ⁇ phase structure in bilayers composed of DOPC:DOPE:DOPS:Chol and can act in synergy with low levels of the cationic lipid DODAC to trigger H ⁇ phase formation.
• Ca 2+ plays a role in mediating endosomal release during calcium phosphate (CaPj) mediated transfection (Loyter, et ah, Proc. Natl. Acad. Sci. USA 79:422-426 (1982); Orrantia, et ah, Experimental Cell Research 190:170-174 (1990)), as well as in polycation- mediated gene transfer (Bottger, et al, Biochimica et Biophysica Acta 1395:78-87 (1998); Haberland, etal, Biochimica et Biophysica Acta 1445:21-30 (1999)).
• CaPj calcium phosphate
• the surprising aspect is that the increased transfection potency of SPLP in the presence of Ca 2+ could not be related to increased uptake of SPLP by the cells, whereas Ca 2+ caused at least a 2-fold increase in uptake of complexes as evidenced by uptake of both lipid and plasmid (Lam, et ah, Biochim Biophys Acta 1463 :279-290 (2000)). It is likely that this discrepancy is related to the much different physical properties of SPLP as compared to complexes. Complexes are large, positively charged systems containing high (equimolar) levels of cationic lipid, whereas SPLP are small, stable, essentially neutral vesicles with a PEG coating that contain low levels of cationic lipid.
• the low levels of cationic lipid in SPLP as compared to complexes may be directly related to enhanced sensitivity to Ca + , as the cationic lipid present in the SPLP may be insufficient to combine with all available anionic lipid in the endosome, thus requiring the additional presence of Ca 2+ to achieve maximum destabilization.
• a local increase in calcium concentration can be produced at the site of transfection, e.g., by local (e.g., intratumoral) delivery of the SPLP along with a high concentration of calcium, or by systemic delivery of the SPLP combined with local delivery of calcium to the desired site of transfection.
• xamp e a i ize asm - ⁇ ia ar ic es on aining a ionic ipi s x i i
• DOPE was obtained from Northern Lipids Inc. (Vancouver, BC). Rh-PE, and PicoGreen were obtained from Molecular Probes (Eugene, OR). DODAC was synthesized and supplied by Dr. S. Ansell of Inex Pharmaceuticals (Vancouver, BC). PEG-CerC o was synthesized as indicated elsewhere (Webb, et ah, Biochim. Biophys. Acta 1372:272-282 (1998)) and was supplied by Dr. Z. Wang of Inex Pharmaceuticals (Vancouver, BC). The pCMVLuc plasmid encodes the Photinus pyralis luciferase gene under the control of the human CMV early promoter and was supplied by Dr. P.
• the pCMVGFP plasmid contains the gene for the green fluorescent protein from Aequorea victoria and was supplied by Dr. P. Tam of Inex Pharmaceuticals (Vancouver, BC).
• DEAE-Sepharose CL-6B, Sepharose CL-4B, octyl- ⁇ -D- galactoside, and HEPES were obtained from Sigma- Aldrich (Oakville, ON).
• Lipofectin was obtained from Gibco BRL (Burlington, ON).
• BHK cells were obtained from Dr. R. MacGillivray of the Department of Biochemistry and Molecular Biology, UBC.
• SPLP-CPL 4 SPLP composed of DOPE:DODAC:PEG-CerC 2 o (84:6:10) and containing the plasmid pCMVLuc (or pCMVGFP) were prepared according to the method of Wheeler, et a (Gene Therapy 6:271- 281 (1999)) using purification by anion exchange (DEAE-Sepharose CL-6B) chromatography and sucrose density gradient centrifugation to remove unencapsulated plasmid and empty vesicles, respectively.
• DEAE-Sepharose CL-6B anion exchange
• SPLP containing Rh-PE were prepared by dissolving Rh-PE with other component lipids in CHC1 3 at a molar ratio of 83.5:10:6:0.5 (DOPE:DODAC:PEG-CerC 20 :Rh-PE) prior to forming the lipid film.
• CPL was inserted into preformed SPLP by incubating SPLP (500 mnol lipid) with CPL 4 (12.5, 19, and 30 nmol) at 60°C for 2 to 3 h in Hepes buffered saline (HBS), pH 7.5, unless otherwise indicated. Unincorporated CPL 4 was removed by gel filtration chromatography on a Sepharose CL-4B column equilibrated in HBS.
• phospholipid was determined using the method of Fiske-Subbarow (J Biol. Chem. 66:375-400 (1925)) following lipid extraction according to Bligh and Dyer (Can. J. Biochem. Physiol. 37:911-917 (1959)). Plasmid DNA was determined using the PicoGreen Assay kit (Molecular Probes, Eugene, Oregon) as previously described (Mok, et al, Biochim. Biophys.
• Lipoplexes were prepared at a charge ratio of 1.5:1 (positive-to-negative) by adding 25 ⁇ L of 88 ⁇ g/mL plasmid DNA (pCMVLuc or pCMVGFP) with 25 ⁇ L of
• DOPE:DODAC 0.8 mM
• Quasi-elastic light scattering (QELS) studies were conducted employing a Nicomp Model 270 Submicron Particle Sizer operating in the vesicle mode. Freeze-fracture electron microscopy studies were performed as described by Wheeler et al , supra.
• DNA for Southern analysis was extracted using a phenol: chloroform extraction following incubation of SPLP systems with 50% mouse serum. The resulting DNA was then subjected to electrophoresis tlirough a 1% agarose gel, transferred to a nylon membrane (Amersham) and subjected to Southern analysis. The membrane was exposed to random-primed 32 P-labelled PvuII restriction fragment from the luciferase gene according to current protocols. Hybridization intensities were quantified using a Phosphorimager SI from Molecular Dynamics. The data were converted to give amounts of intact DNA relative to undigested DNA.
• Plasmid DNA concentrations corresponded to 1.4 ⁇ g/mL and 2.2 ⁇ g/mL for the SPLP systems and the lipoplexes, respectively.
• Cells were incubated at 37°C for indicated periods, washed twice with PBS and lysed with 600 ⁇ L of lysis buffer (0.1% Triton X-100 in PBS).
• Rhodamine fluorescence was determined using a ⁇ ex of 560 nm and a ⁇ em of 600 nm with slit widths of 10 and 20 nm, respectively. An emission filter of 530 nm was also used.
• Lipid uptake was determined by comparison of the fluorescence in the lysate to that of a lipid standard and normalized to the cell number as determined by the BCA protein assay (Pierce, Rockford, IL). Where indicated, fluorescence micrographs were obtained using an Axiovert 100 Zeiss Fluorescent microscope (Carl Zeiss Jena GmbH) using a rhodamine filter from Omega Opticals (Brattleboro, VT) with the following specifications: excitation 560+20/dichroic filter 590/long pass emission 600. The effect of Ca 2+ and Mg 2+ on lipid uptake was determined as described above with the following exceptions.
• BHK cells (5x10 4 per well) were seeded in a 24-well plate in 1 mL of complete media and incubated overnight at 37°C.
• SPLP-CPL 4 (40 nmol) were mixed with CaCl 2 or MgCl 2 in a total volume of 100 ⁇ L.
• Complete media 400 ⁇ L was added to the SPLP-CPL resulting in final cation concentrations of 4 to 14 mM. This mixture was then added to the cells and incubated for 4 h at 37°C. Cells were then washed twice with PBS and lysed in 600 ⁇ L of lysis buffer (0.1% Triton X-100 in PBS).
• transfection studies were performed employing lxl 0 4 BHK cells plated in each well of a 96-well plate in 150 ⁇ L complete media prior to overnight incubation at 37°C in 5% CO 2 .
• SPLP and SPLP-CPL corresponding to 0.5 ⁇ g of pCMVLuc in 20 ⁇ L HBS (SPLP), or HBS containing 40 mM CaCl 2 (SPLP-CPL 4 ) were added to 80 ⁇ L of complete media for a plasmid concentration of 5.0 ⁇ g/mL.
• a transfection time of 4 h with a total incubation time of 24 h was used routinely.
• the transfection time is defined as the time the cells are incubated with the plasmid-containing particles whereas the total incubation time is the transfection time (after which the transfection media is replaced) plus the subsequent time the cells are incubated for prior to assaying for transgene expression.
• the cells were lysed with 100 ⁇ L of lysis buffer, and 40 ⁇ L of the lysate was transferred to a 96-well luminescence plate.
• Luciferase activity was determined using a Luciferase reaction kit (Promega, Madison, WI), a luciferase standard (Boehringer- an eim , an a micro i er p a e ummome er om o ecu ar ynamics (Chantilly, VA). Activity was normalized to the number of cells as determined by the BCA protein assay (Pierce, Rockford, IL).
• the transfection time course study included SPLP, SPLP-CPL, and Lipofectin (Gibco BRL, Burlington, ON) and DOPE/DODAC lipoplexes containing pCMVLuc.
• the lipoplexes were prepared as described earlier. After transfection times of 4, 8, and 24 h the transfection media was removed and in the case of the 4 and 8 h transfections, was replaced with complete media for a total incubation time of 24 h. At 24 h, all cells were lysed and assayed for luciferase activity and protein content (BCA assay), as described above.
• SPLP-CPL DOPE:DODAC lipoplexes and Lipofectin lipoplexes containing pCMVGFP were prepared as described for pCMVLuc.
• the transfections were performed as described above at a plasmid DNA dose of 5.0 ⁇ g/mL. Following incubation of the samples for 24 and 48 h, the transfection media was removed, the cells were washed, and fresh media was added to the cells. The cells were then viewed under a Zeiss fluorescence microscope. The number of cells expressing GFP were counted using a fluorescein filter (Omega).
• Opticals with the following specifications: excitation 475+20/dichroic filter 500/emission 535+22.5.
• the transfection efficiency was expressed as percentage of cells expressing GFP.
• Cationic PEG lipids can be inserted into preformed SPLP. Previous work has shown that SPLP exhibit lower uptake into cells and lower transfection potencies than lipoplexes (Mok, et ah, Biochim. Biophys. Acta 1419:137-150 (1999)). It has also been shown that surface-associated cationic PEG lipids (CPL), particularly those containing four charges at the end of the PEG molecule (CPL ; for structure see Figure 8 A), can dramatically enhance the uptake of LUV into cells. Further, CPL can be inserted into preformed LUV with lipid compositions similar to SPLP employing a straightforward incubation protocol. It was thus examined whether a similar procedure could be developed to insert CPL 4 into SPLP.
• CPL surface-associated cationic PEG lipids
• SPLP containing pCMVLuc were prepared by the detergent dialysis procedure of Wheeler et al. (Gene Therapy 6:271-281 (1999)) from a lipid mixture containing 6 mol% of the cationic lipid N,N-dioleoyl-N,N-dimethyl ammonium chloride (DODAC), 84 mol% of the "fusogenic" helper lipid dioleoyl phosphatidylethanolamine (DOPE) and 10 mol%> of a stabilizing lipid consisting of PEG 2 ooo attached to a ceramide (Cer) anchor (PEG-Cer).
• DODAC cationic lipid N,N-dioleoyl-N,N-dimethyl ammonium chloride
• DOPE "fusogenic" helper lipid dioleoyl phosphatidylethanolamine
• PEG-Cer ceramide
• the ceramide anchor of the PEG-Cer contained a C o acyl chain (PEG-CerC o) that does not readily exchange out of the vesicle, thus contributing to a highly stable SPLP system (Wheeler, et al, Gene Therapy 6:271-281 (1999)).
• the detergent dialysis procedure results in the formation of a mixture of SPLP containing one plasmid per vesicle, free plasmid, and empty vesicles. SPLP were purified by removing free plasmid and empty vesicles by DEAE column chromatography and density centrifugation, respectively, as described elsewhere (Wheeler, et al. , Gene Therapy 6:271-281 (1999)).
• SPLP-CPL 4 aggregate following insertion of CPL 4 and de- aggregate following addition of divalent cations. Previous work has shown that LUV containing CPL tend to aggregate and that this aggregation can be inhibited by increasing the ionic strength of the medium. It was found that SPLP-CPL 4 were also susceptible to aggregation, and that this aggregation could be reversed by adding NaCl, CaCl 2 or MgCl 2 to the SPLP-CPL formulation. This effect is illustrated in Figure 10 which shows the effect of the addition of CaCl 2 and MgCl 2 on aggregation of SPLP-CPL 4 as monitored by the change in the standard deviation of the mean diameter of the particles measured by quasi-elastic light scattering (QELS).
• QELS quasi-elastic light scattering
• the sizes of SPLP and SPLP-CPL 4 in the presence of CaCl 2 were compared using QELS and freeze-fracture electron microscopy.
• QELS studies revealed the mean diameter of SPLP and SPLP-CPL 4 to be 80 + 19 nm and 76 + 15 nm, respectively, whereas the freeze-fracture studies indicated diameters of 68 ⁇ 11 nm and 64 + 14 nm. These values for SPLP diameters are in close agreement with previous studies (Wheeler, et al, Gene Therapy 6:271-281 (1999)).
• PEG-CerC 2 o content and stability of SPLP-CPL 4 .
• CPL 4 can be inserted to achieve levels as high as 4 mol% of the total SPLP lipid indicates that the level of CPL 4 in the outer monolayer of the SPLP-CPL is 8 mol%. Given that the initial concentration of PEG-CerC 0 is 10 mol%, this suggests that the total levels of PEG-lipids in the outer monolayer of the SPLP-CPL 4 can approach 18 mol%. These levels are higher than the levels of PEG-lipids that can usually be incorporated into lipid vesicles (Woodle, et ah, Biochim. Biophys.
• the PEG-CerC 2 o-to-DOPE ratio Prior to insertion of the CPL 4 the PEG-CerC 2 o-to-DOPE ratio was 0.091, corresponding to a PEG-CerC 2 o content of 7.6 mol%, assuming that the DOPE constituted 84 mol% of the lipid content. Following insertion of the CPL 4 the PEG-CerC 2 o-to-DOPE ratio was found to be 0.072, indicating a PEG-CerC 20 content of 6.0 mol%.
• SPLP-CPL 4 exhibit enhanced uptake into BHK cells and dramatically enhanced transfection potency.
• the next set of experiments was aimed at determining the influence of incorporated CPL 4 on the uptake of SPLP into BHK cells and the resulting transfection potency of the SPLP-CPL 4 system.
• SPLP containing up to 4 mol% CPL 4 were prepared in the presence of 40 mM CaCl 2 and were added to BHK cells (final CaCl 2 concentration 8 mM) and incubated for varying times. The cells were then assayed for associated SPLP-CPL 4 as indicated in the above Materials and Methods.
• the transfection properties of SPLP, SPLP-CPL 4 and plasmid DNA-cationic liposome lipoplexes were examined using the transfection protocol described in the above Materials and Methods.
• This protocol involves incubation of BHK cells with SPLP or lipoplexes for 4 h (the transfection time) followed by replacement of media and further incubation to maximize transgene expression.
• the total incubation time was kept constant at 24 h.
• the presence of increasing amounts of CPL 4 resulted in dramatic increases in the transfection potency for the SPLP system.
• SPLP-CPL 4 containing 4 mol% CPL 4 exhibited luciferase expression levels some 3xl0 3 higher than achieved with SPLP.
• Ca 2+ is required for transfection activity of SPLP-CPL 4 .
• Example I demonstrates that the transfection potency of SPLP is highly sensitive to the presence of Ca 2+ , where the presence of ⁇ 10 mM Ca 2+ enhances transfection potency several hundred-fold. It was therefore of interest to determine the influence of Ca 2+ on the transfection activity of SPLP-CPL 4 .
• SPLP containing 4 mol% CPL were incubated with BHK cells for 48 h in the presence of varying amounts of MgCl 2 and CaCl , and the luciferase activities were determined. As shown in Figure 15, the transfection activity was primarily dependent on the presence of Ca 2+ in the transfection medium.
• SPLP-CPL 4 exhibited transfection potencies that were more than 10 5 times higher than if the corresponding amount of MgCl 2 was present. In order to determine whether the different transfection properties of SPLP-
• SPLP-CPL 4 exhibit transfection potencies in vitro that are comparable to or greater than the transfection potencies of lipoplexes.
• the data presented in Figure 14 indicate that DOPE/DODAC lipoplexes yield ⁇ - 100-fold higher levels of gene expression than SPLP-CPL 4 when applied to BHK cells for a period of 4 h. Given that SPLP-CPL 4 are stable systems, uptake can conceivably continue over extended time periods. The transfection levels achieved when SPLP-CPL 4 or the lipoplexes were applied to BHK cells for transfection times of 4, 8 and 24 h were thus examined.
• DOPE:DODAC (1:1) lipoplexes charge ratio 1.5
• lipoplexes generated using the transfection reagent Lipofectin consisting of DOPE/DOTMA (1:1) lipoplexes at a charge ratio of 1.5.
• the potency of SPLP-CPL 4 increases markedly with increased transfection times, suggesting that the rate of uptake of the SPLP-CPL 4 system may be a limiting factor for transfection.
• transfection levels are comparable to those achieved by Lipofectin or the DOPE/DODAC lipoplexes.
• SPLP-CPL 4 are nontoxic and efficient transfection agents. It is well known that lipoplexes can be toxic to cells. The SPLP-CPL 4 contain low levels of cationic lipid and are potentially less toxic than lipoplexes. The toxicities of SPLP-CPL and lipoplexes were assayed by determining cell viability following a 24 h and 48 h exposure to levels of SPLP-CPL 4 and lipoplexes corresponding to 5.0 ⁇ g/mL plasmid, corresponding to total lipid doses of approximately 80 ⁇ M and 45 ⁇ M for SPLP-CPL and lipoplexes, respectively. As shown in Figure 18B, SPLP-CPL 4 exhibited little toxicity, whereas lipoplexes were highly toxic. Cell survival was only 30% after a 48 h incubation with Lipofectin lipoplexes, whereas -95% of the cells were viable following a 48 h incubation with SPLP-CPL 4 .
• cryo-electron microscopy studies which indicate that structural changes (from spheres to discs) are only observed for distearoylphosphatidylcholine (DSPC) liposomes at PEG-PE levels above 12 mol%, with lytic effects observed above 15 mol% (Edwards, et al. , Biophys. J. 73 :258-266 (1997)).
• X-ray studies indicate that nonbilayer micellar structures are only observed for PEG-lipid levels above 15 mol% (Kenworthy, et al, Biophys. J. 68:1903-1920 (1995); Hristova, etal, Macromolecules 28:7693-7699 (1995)).
• the aggregation phenomenon may be related to the ability of PEG coatings to adopt a conformation that is able to bind proteins such as streptavidin (Sheth, et al, Proc. Natl. Acad. Sci. USA 94:8399-8404 (1997)).
• streptavidin Sheth, et al, Proc. Natl. Acad. Sci. USA 94:8399-8404 (1997).
• the second point of discussion concerns the mechanism whereby CPL 4 increases the transfection potency of the SPLP system.
• the transfection potency of SPLP-CPL 4 (in the presence of Ca 2+ ) is increased by a factor of -10 4 (Figure 14) in comparison to the transfection potency of SPLP in the absence of Ca 2+ .
• SPLP-CPL are small, homogeneous, stable systems containing one plasmid per particle (Wheeler, et al, Gene
• SPLP are basic components of more sophisticated systems, such as SPLP-CPL 4 , that can be constructed in a modular fashion. For example, post-insertion of PEG-lipids containing specific targeting ligands in place of the cationic groups of CPL should result in SPLP that are specifically targeted to particular cells and tissues. With regard to toxicity, SPLP-CPL 4 are markedly less toxic to BHK cells in tissue culture than are lipoplexes.

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