KR100650959B1 - Condensed plasmid-liposome complex for transfection - Google Patents

Abstract

The present invention relates to plasmid-liposomal compositions for transfection of cells. The composition comprises plasmid molecules and cationic liposomes condensed with a polycationic condensing agent. Also described are methods of making plasmid-liposomal complexes.

Plasmid-Liposomes, Polycationic Condensers, Transfection

Description

Condensation plasmid-liposomal complex for transfection {CONDENSED PLASMID-LIPOSOME COMPLEX FOR TRANSFECTION}

1 is a computer image of electron micrographs of plasmids condensed with histones throughout the polycationic polymer.

FIG. 2 is a computer image of electron micrographs of plasmids condensed with polycations, complexed with cationic liposomes according to the present invention.

3A-3B are computer images of cryogenic (FIG. 3A) and freeze fracture (FIG. 3B) transmission electron micrographs of plasmids condensed with polycations complexed with cationic liposomes according to the present invention. .

Figure 4 is a view showing the size (nm) when measured by dynamic light scattering (dynamic light scattering) of the plasmid-liposomal complex prepared according to the present invention.

FIG. 5 is a plot of particle diameter (nm) measured by dynamic light scattering as a function of storage time at 4 ° C. for the plasmid-liposomal complex of the present invention. FIG.

FIG. 6 is a plot of the amount of injection as a function of time after intravenous intravenous injection of a labeled plasmid-liposomal complex of the present invention.

7A-7E show luciferase in the lung (FIG. 7A), liver (FIG. 7B), heart (FIG. 7C), spleen (FIG. 7D) and kidney (FIG. 7E) for plasmid-liposomal complexes prepared with whole histones. Expression (relative light unit (RLU) / 10 sec / mg protein).

8A-8E show luciferase in the lung (FIG. 8A), liver (FIG. 8B), heart (FIG. 8C), spleen (FIG. 8D) and kidney (FIG. 8E) for plasmid-liposomal complexes prepared with histone H1. Expression (relative light unit (RLU) / 10 sec / mg protein).

9A-9E are luciferases in the lung (FIG. 9A), liver (FIG. 9B), heart (FIG. 9C), spleen (FIG. 9D) and kidney (FIG. 9E) for plasmid-liposomal complexes prepared with histone H4. Expression (relative light unit (RLU) / 10 sec / mg protein).

10A-10E show lung (FIG. 10A), liver (FIG. 10B), heart (FIG. 10C), spleen for plasmid-liposomal complexes prepared with poly-glutamine, melittin or polymyxin B as cationic condensing agents. Luciferase expression (pg luciferase / mg protein) in (FIG. 10D) and kidney (FIG. 10E).

11A-11B show luciferase expression in lungs (FIG. 11A) and liver (FIG. 11B) as a function of time (days) for plasmid-liposomal complexes stored at 4 ° C. FIG.

FIG. 12 shows luciferase (pg / mg protein) in various tissues 24 hours after intravenous intravenous administration of plasmid-liposomal complexes as a function of micrograms of luciferase bearing plasmids.

FIG. 13 shows luciferase activity (RLU / mg protein) in various tissues 24 hours after intravenous administration of plasmid-liposomal complexes.

Figures 14A-14B show surviving animals as a function of days after inoculation of Lewis lungs prior to Lewis lung for animals treated with plasmid-liposomal complexes comprising plasmids pHSVtk (FIG. 14A), pCMVp53 (FIG. 14B) and pCMVIL2 (FIG. 14C). Indicates a percentage.

FIG. 15 shows the body weight (g) of mice after administration of plasmid-liposomal complexes prepared with pNSL plasmids encoding luciferase as a function of time in days. Mice were challenged with plasmid-liposomal complexes at doses of saline (+), and 50 μg plasmid (O), 75 μg plasmid (▲) and 100 μg plasmid (▽), as indicated by the arrows in the figure. Treatments were taken on days 22 and 28.

FIG. 16 shows luciferase expression (ng / mg) in various mouse tissues 24 h after administration of plasmid-liposomal complexes prepared with luciferase-encoding pNSL plasmids to tumor-bearing mice (metastatic models of Lewis lung tumors). ).

The present invention relates to an improved method for preparing a plasmid-liposomal complex for in vivo transfection of a gene and to a composition prepared by the method.

references

Felgner, J., et al., Proc. Natl. Acad Sci. USA 84 : 7413-7417 (1987).

Felgner, J., et al., J. Tiss. Cult. Meth. 15 : 63-68 (1993).

Gao, X., and Huang, L., Biochemistry 35 : 1027-1036 (1996).

Guo, L., et al., Journal of Liposome Research 3 (1): 51-70 (1993).

Li, S., and Huang, L., Journal of Liposome Research, 6 (3): 589-608 (1996).

Mulligan, R. S., Science 260: 926-932 (1993).

Morishita, R., et al., J. Clin. Invest. 91: 2580-2585 (1993).

Rose, J.K., U.S. Patent No. 5,279,833 (1994).

Trubetskoy, VS, et al., Biochimica et Biophysica Acta 1131 : 311-313 (1992).

Wagner, E., et al., Proc. Natl. Acad Sci. USA 88: 4255-4259 (1991).

Various methods have been developed to facilitate the delivery of genetic material (eg, gene therapy) to specific cells. These methods are useful for in vivo or ex vivo gene delivery. In the former, the gene is introduced directly into the subject (intravenous, intraperitoneal, aerosol, etc.). In ex vivo (or in vitro) gene delivery, a gene isolates a cell from a particular tissue of the subject and then introduces it into the cell, and then introduces the transfected cell back into the subject.

Delivery systems for in vivo and ex vivo gene therapy include viral vectors such as retroviral vectors or adenovirus vectors, microinjection, electroporation, protoplast fusion, calcium phosphate and liposomes (Felgner, et al., 1987; Mulligan, 1993; Morishita et al., 1993),

For example, liposome mediated gene therapy involves the use of cationic liposomes formed from LIPOFECTIN, a reagent consisting of cationic lipids and neutral lipids (Felgner, et al., 1989, 1993). Other liposome mediated methods of gene therapy, which form electrostatic complexes of cationic liposomes and DNA, have been described (Trubetskoy, et al., 1992; Morishita et al., 1993; Rose, 1994). More recent approaches to liposome based transfection compositions include polyvalent binding of DNA to lipid particles (Wagner, et al., 1991) or condensation of DNA (Gao and Huang, 1996; Li and Huang, 1996). Cations such as protamine or polylysine.

However, liposome mediated gene therapies and compositions described to date have limitations, including, for example, the toxicity of LIPOFECTIN, the large size of the DNA-liposomal complex, and rather poor in vivo transfection efficiency.

Thus, it would be desirable to prepare DNA plasmid-liposomal complexes that are relatively nontoxic, have an intravenous size, and have high transfection efficiency.

In one embodiment, the present invention includes compositions of plasmid-liposomal complexes used to transfect host cells with genes contained in plasmids. The composition includes plasmid molecules condensed with a polycationic condensing agent and suspended in an aqueous medium of low ionic strength, and cationic liposomes formed from cationic vesicle forming lipids. This complex has a ratio of liposome lipid to plasmid greater than 5 nmole of liposome lipid / plasmid to less than 25 nmole of liposome lipid / plasmid and having a substantially uniform size of less than about 200 nm.

In one embodiment, the condensed plasmid molecule is selected from the group consisting of cystic fibrosis transmembrane conductance regulators, genes encoding Factor 8 (Factor VIII), interleukin-2 or p53. DNA plasmid molecules containing genes.

In one embodiment, the condensing agent is a polycation selected from histones, poly-1-glutamine, protamine, melittin and polymyxin B. In a preferred embodiment, the condensing agent is a histone selected from total histone, histone 1 and histone 4.

In another embodiment, the ratio of liposome lipid to plasmid is μg of liposome lipid / plasmid of 8-18 nmoles.

Low ionic strength aqueous media are prepared from nonionic osmotic solutes such as glucose, sucrose or dextran.

Cationic liposomes include dimethyldioctadecylammonium (DDAB), 1,2-dioleyloxy-3- (trimethylamino) propane (DOTAP), N- [1- (2,3-ditetradecyloxy) propyl] -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N- [1- (2,3-dioleyloxy) propyl] -N, N-dimethyl-N-hydroxy ethylammonium bromide (DORIE ), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) and 3β [N- (N ', N'-dimethylaminoethane) carba Mole] cholesterol (DC-Chol) consists of cationic vesicle forming lipids.

In another embodiment, the cationic liposomes further comprise neutral vesicle forming lipids. In another embodiment, the cationic liposomes further comprise cholesterol.

In another embodiment, the cationic liposomes are surface coated with hydrophilic polymer chains by including vesicle forming lipids derived from hydrophilic polymers. In one embodiment, at least a portion of the hydrophilic polymer chains are bound to the endoplasmic reticulum by binding that is effective to release the hydrophilic polymer chains in response to release bonds, such as existing or induced physiological conditions. The plasmid-liposomal complex may further comprise a ligand attached to the end of the hydrophilic polymer chain for ligand specific binding to the receptor molecule on the target cell surface. For example, in a preferred embodiment, the hydrophilic polymer is polyethylene glycol.

In another aspect, the invention provides a method for the preparation of a plasmid-liposomal complex that condenses plasmid molecules and mixes the condensed plasmid with a suspension of cationic liposomes to form plasmid-lipid complexes used for transfection of host cells. Improvements in the manufacturing method are included. This improvement includes (i) the selection of a polycation selected from histones, poly-glutamine, protamine, melittin and polymyxin B as condensing agents for condensation of plasmid molecules, and (ii) as a suspending medium for condensed plasmid molecules. , Selection of an aqueous medium of low ionic strength, and (iii) selection of liposome lipid to plasmid ratio of greater than 5 nmole of liposome lipid / plasmid to less than 25 nmole of liposome lipid / plasmid. The plasmid-liposomal complexes prepared by this improvement have a substantially uniform size of less than 200 nm.

Plasmid-liposomal complexes prepared according to the methods of the invention, in one embodiment, are used to transfect host cells with the genes contained in the DNA plasmid, which DNA plasmid is factor 8, interleukin-2 or p53. It includes a gene selected from the group consisting of the gene encoding the.

In a preferred embodiment, the plasmid-liposomal complex is a cystic fibrosis transmembrane conductance regulator, or a DNA plasmid containing a cytokine such as interleukin-2 for lung cancer, or a tumor suppressor gene such as p53, to target a host cell in the lung of the subject. Used to transfect.

These and other objects and features of the present invention will become more apparent upon reading the following detailed description of the invention in conjunction with the accompanying drawings.

I. Plasmid- Liposome Complex

The present invention is directed to plasmid-liposomal complexes and improvements in methods of making such complexes for use in in vivo transfection of host cells. The composition comprises plasmid molecules suspended after condensation with a polycationic condensate in a medium of low ionic strength. The condensed plasmid molecules are mixed with lipid particles such as liposomes to form plasmid-liposomal complexes. Improvements in the method of preparation, as described below, involve the selection of condensing agents, the choice of suspension media and the selection of liposome lipid to plasmid ratios. Before detailing the improved preparation procedure, the plasmid-liposomes and components thereof are described.

A. Cationic Liposomal Ingredients and Preparation

As noted above, plasmid-liposomal complexes comprise condensed plasmid molecules and liposomes. Liposomes as used herein refers to lipid vesicles having an outer lipid shell formed of one or more lipid bilayers, typically encapsulating an aqueous interior. In a preferred embodiment, the liposome is a cationic liposome consisting of about 20 to 80 mole percent of the cationic vesicle forming lipids and the remainder neutral vesicle forming lipids and / or other components. As used herein, an "vesicle forming lipid" is any amphiphilic lipid having hydrophobic and polar head group moieties, as exemplified by phospholipids, that can itself form spontaneously bilayer vesicles in water. Means. Preferred vesicle-forming lipids are diacyl chain lipids, such as phospholipids, the acyl chains of which are typically about 14 to 22 carbon atoms in length and have varying degrees of unsaturation.

Cationic vesicle forming lipids are lipids in which the polar head group has a net positive charge at an operating pH such as pH 4-9. Typical examples include, for example, phosphatidyl, wherein a polar hair group is induced with a positive moiety (eg lysine), as described for _L -lysine-induced lipid DOPE (LYS-DOPE) (Guo, et al., 1993). Phospholipids such as ethanolamine. This class also includes glycolipids such as cerebrosides and gangliosides having cationic polar headgroups.

Other cationic vesicle forming lipids that can be used are cholesterol amines and related cationic sterols. Examples of cationic lipids include 1,2-dioleyloxy-3- (trimethylamino) propane (DOTAP), N- [1- (2,3-ditetradecyloxy) propyl] -N, N-dimethyl- N-hydroxyethylammonium bromide (DMRIE), N- [1- (2,3-dioleyloxy) propyl] -N, N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), N- [1 -(2,3-Dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), 3β [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol ) And dimethyl dioctadecyl ammonium (DDAB).

The remainder of the liposomes is formed of neutral vesicle forming lipids, meaning vesicle forming lipids that may contain small amounts of lipids that do not have a net charge or have a negative charge on the polar head group. Lipids of this class include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and sphingomyelin (SM) and cholesterol, cholesterol derivatives and other non-charged sterols.

The aforementioned lipids can be purchased commercially or prepared according to known methods. Other lipids that may be included in the present invention are glycolipids such as cerebroside and ganglioside.

In one embodiment of the present invention, the plasmid-liposomal complex includes liposomes surface-coated with hydrophilic polymer chains that are effective for prolonging the blood circulation time of the plasmid / liposomal complex. Suitable hydrophilic polymers include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinyl-pyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylic Amides, and derived celluloses such as hydroxymethylcellulose or hydroxyethylcellulose. Preferred hydrophilic polymer chains are polyethylene glycol (PEG), preferably PEG chains having a molecular weight of 500-10,000 Daltons, more preferably 1,000-5,000 Daltons. Hydrophilic polymers can be dissolved in non-aqueous solvents such as water and chloroform.

The coating is preferably prepared by incorporating 1 to 2 mole percent of the endoplasmic lipid forming phospholipid or other diacyl-chain lipids, preferably in the vesicle-forming lipid forming liposomes, preferably having a polymer chain in the head group. Methods of making such lipids and thus exemplary methods of forming polymer coated liposomes are described in US Pat. Nos. 5,013,556 and 5,395,619.

Hydrophilic polymers may be stably bound to lipids, or may be bound via labile bonds that cause the polymer coated plasmid liposome complexes to drop or release the hydrophilic polymer coating, either during circulation in the bloodstream or after settling at the target site. You will know. The attachment of hydrophilic polymers, in particular polyethylene glycol (PEG), to endoplasmic forming lipids through binding effective to release the polymer chains in response to stimulation is described, for example, in WO 98/16202 and WO 98/16201, and Kirpotin, D. et al., FEBS Letters, 388: 115-118 (1996).

In one embodiment, the releasing bond is a chemical releasing bond that is cleaved by administration of the appropriate releasing agent or under selective physiological conditions such as the presence of an enzyme or reducing agent. For example, ester and peptide bonds are cleaved by esterase or peptidase enzymes. Disulfide bonds are cleaved by the administration of a reducing agent such as glutathione or ascorbate or by a reducing agent present in vivo such as plasma and intracellular cysteine.

Other release bonds include pH sensitive bonds, and bonds that are cleaved upon exposure to glucose, light or heat. For example, hydrophilic polymer chains can be attached to liposomes by pH sensitive bonds, and plasmid-liposomal complexes target sites, such as tumor regions, having a pH effective to cleave bonds and release hydrophilic chains. Examples of pH sensitive bonds include acyloxyalkyl ethers, acetals and ketal bonds. Another example is when the cleavable bond is a disulfide bond, which is meant herein to be a sulfur containing bond inclusive. Sulfur containing bonds may be synthesized to achieve the selected degree of instability and may include disulfide bonds, mixed sulfide-sulfone bonds and sulfide-sulfoxide bonds. Of these three bonds, disulfide bonds are least sensitive to thiolysis and sulfide-sulfoxide bonds are most sensitive.

Such release bonds are useful for controlling the release rate of hydrophilic polymer fragments from plasmid-liposomal complexes. For example, very unstable sulfide bonds can be used for targeting to blood cells or endothelial cells because these cells are easy to access and the short blood circulation life of the liposomes is sufficient. Conversely, long-lasting or strong disulfide binding can be used when the target is tumor tissue, or other organ that generally requires a long liposome circulating life to reach the desired target.

Release bonds that attach the hydrophilic polymer chain to the liposomes are typically the result of changes in the environment, such as in certain areas with slightly lower pH, such as areas of tumor tissue, or sites with reducing conditions, such as hypoxic tumors. If so, it is cleaved in vivo. In vivo reducing conditions may also be achieved by the administration of a reducing agent such as ascorbate, cysteine or glutathione. Cleavable bonds can also be cleaved in response to external stimuli such as light or heat.

In another embodiment, the plasmid-liposomal complexes comprise an affinity moiety or targeting ligand that is effective for specifically binding to the target cell to be treated. Such moieties may be attached to the surface of liposomes or attached to the ends of the hydrophilic polymer chains. Examples of such moieties include antibodies, such as those described in PCT applications WO US94 / 03103, WO98 / 16202 and WO98 / 16201, ligands that specifically bind to target cell surface receptors, and the like. The moiety may also be a hydrophobic fragment that promotes fusion of the complex with the target cell.

B. Condensed Plasmids

This section describes the preparation of condensed-phase plasmids for use in the plasmid-liposomal complexes of the present invention.

Polycationic condensing agents used to condense plasmids are multicharged cationic polymers, typically biopolymers such as spermidine, spermine, polylysine, protamine, whole histones, certain histone fragments such as H1, H2, H3 , H4 and other polycationic polypeptides, but may also include biocompatible polymers such as polymyxin B. Such polycationic condensing agents may also be used in the form of free bases or salts, such as protamine sulfate and polylysine hydrobromide. In a preferred embodiment, the polycationic condensing agent is histone, which includes whole histones or specific histone fragments as mentioned herein.

Plasmids suitable for use in the complex are preferably cyclic or closed double-stranded molecules having a size in the range of 5-40 Kbp (kilo basepair). This plasmid is constructed in accordance with known methods and is expressed during gene therapy under the control of therapeutic genes, ie, suitable promoter and terminator control elements and other elements necessary for replication and / or integration into the host cell genome in the host cell. Contains the genes. Methods of making plasmids useful for gene therapy in genes or other mammals are well known and referenced.

Genes introduced for gene therapy by the complexes of the invention typically fall into one of three categories:

The first is a gene for overcoming a deficiency or defect in a gene in a subject. That is, when the subject fails to produce active endogenous proteins at all or within normal levels, the genes introduced into the plasmids are intended to compensate for this deficiency. Examples of this class of genes include genes encoding adenosine deaminase (ADA) for gene expression in stem cells or lymphocytes; Genes encoding treatment of Lesch-Nyhan syndrome caused by purine nucleoside phosphorylase deficiency, deficiency of prostaglandin G / H synthase, deficiency of hypoxanthine-guanine phosphoribosyltransferase, hemophilia, sickle anemia And various circulating proteins such as α ₁ -antitrypsin, coagulation factors (eg, factor 8, ninth), and globins (eg, β-globin, hemoglobin) for the treatment of other blood-related diseases. Genes and genes encoding hormones and other peptide modulators.

In the second class, polypeptides are designed to treat any existing pathology, such as a tumor, or a pathogenic condition, such as a viral infection. In this example, the gene for supplying the specific antibody genes to inhibit a pathogen genes, and targeting of the Pseudomonas peptide to inhibit the epidermal cell binding of the gene for the CD4 peptide to inhibit the p53 gene, HIV infection for treatment of tumors, Pseudomonas (Pseudomonas) Treatment is included.

The third class includes genes designed to produce mRNA transcripts that can act as antisense molecules that inhibit undesirable protein expression, such as overexpression of proteins specific for tumor growth or expression of viral proteins.

II. Preparation and Characterization of Composites

According to the invention, plasmid-liposomal complexes formed by mixing condensed plasmids and cationic liposomes in low ionic strength media are typically less than about 200 nm, preferably 50-200 nm, more preferably 100-200 nm. It was found, most preferably, to produce a composite having a substantially uniform size of 120-180 nm. This complex is further characterized by the retention of the activity of the expression of the encoded gene, ie the complex has transfection stability. This is evidenced by the ability of the complex to store cells at 4 ° C. for 30 days, preferably 90 days, as described below, and then transfect cells and realize expression of the encoded genes.

Condensed phase plasmids are formed by adding a polycationic polymer condensing agent of the type described above in a low ionic strength medium to the plasmid. The cationic polymer is added to the plasmid solution at the desired concentration at which charge stoichiometry is achieved. That is, the total number of charges (determined from the known charge density / weight of the polymer) in the cationic polymer is at least as high as the total negative charge of the DNA (determined from the plasmid weight and the known charge density / weight of the DNA). Typically, the weight ratio of added DNA plasmid to added polymer is about 0.1-5.0, more preferably 0.3-2.0. The condensing agent is preferably added slowly to the plasmid suspension with stirring, eg over 10 minutes.

The condensed plasmid and cationic liposomes contained in the low ionic strength medium are then mixed with the slow addition of the condensed plasmid molecules to the liposomes. The ratio of liposome lipids to plasmids is an important variable in achieving maximum transfection. This ratio (μg of liposome lipid / plasmid of nmole) is greater than 5 to less than 25, preferably greater than 8 to less than 18, more preferably greater than 10 to less than 15, most preferably 12 to 14.

As mentioned above, an important feature of the present invention is the mixing of liposomes and condensate plasmids in low ionic strength media. Preferably the final concentration of the medium, including the ions present in the DNA, condensing agent and liposome lipid species, is less than the ionic strength of the 25 mM monovalent ionizable salts (eg sodium chloride), preferably less than 10 mM of such salts, more preferably Is less than about 1 mM. Typically, low ionic strengths use free base or free acid plasmids, polycationic and lipid species, and use components at concentrations sufficiently diluted to remove electrolyte components or otherwise maintain low ionic strengths. Or by removing the electrolyte produced from the constituents by dialysis or the like. At the same time, as an injection, it is useful to prepare the composition in the presence of a non-electrolyte solute such as glucose, which provides an osmotic balance.

1 is a computer image of negative-stain transmission electron micrographs of luciferase encoding pNSL plasmids condensed with whole histones, prepared as described in the Examples. As can be seen, the plasmid condenses into discrete single particles up to about 100 nm in diameter.

FIG. 2 is a computer image of a negative stain transmission electron micrograph of a polycationic condensation plasmid-liposomal complex prepared as described in Example 1. FIG. When comparing the purely condensed plasmid molecule of FIG. 1 with the complex of FIG. 2, an opaque condensed plasmid is readily visible in FIG. 2. Also shown in FIG. 2 is a clear membrane surrounding each condensed plasmid. This clear membrane is a liposome lipid bilayer covering each condensed plasmid.

3A-3B are computer images of cryogenic electrons and freezing end negative stain transmission electron micrographs of plasmid-liposomal complexes prepared as described in Example 1. FIGS. The micrograph of FIG. 3A is obtained by freezing a thin film of the plasmid-liposomal complex suspension and observing the layer under cold author microscopy. The micrograph shows a separate single plasmid-liposomal complex, where the condensed DNA appears as the darker central region of many particles. The freeze cluster micrograph of FIG. 3B shows no clear aggregates and shows a distinct plasmid-liposomal complex.

Dynamic light scattering was used to determine the average complex size and size distribution. This result is shown in FIG. 4 showing that the composite prepared in Example 1 forms a homogeneous population having an average size of about 146 nm (standard deviation 45 nm). The present and other studies conducted to support the present invention are directed to complexes having a homogeneous population that, when prepared according to the described methods, demonstrate that the composition is a single peak representing a population of complexes having a relatively narrow size distribution. Indicates completion. Preferably, the composite has a size of less than 200 nm, more preferably 50-200 nm, most preferably 100-200 nm, even more preferably 120-180 nm.

As discussed briefly above, the plasmid-complex is stable. That is, the complex retains its initial size, such as few aggregates of the complex, and the complex retains therapeutic activity after storage at 4 ° C. for at least 30 days. 5 provides evidence of complex size stability and shows the complex size as a function of time measured by dynamic light scattering. Suspensions of plasmid-liposomal complexes in water / glucose were stored at 4 ° C. and analyzed at 7, 14, 21, 28, 60 and 90 days after storage. The average complex size after complex formation was about 150 nm, and as seen, even after 90 days of storage, the complex size remained at about 150 nm. Complex stability with respect to maintenance of therapeutic activity is discussed in Figures 11a-11b below.

III. In vivo Transfection

Plasmid-liposomal complexes prepared according to the present invention were administered to mice to determine transfection efficiency of various plasmid-liposomal complex preparations, and to measure stability of transfection, in vivo distribution of the complexes, pharmacokinetics and dose response. It was. Exemplary in vivo transfection procedures are described in Example 2.

The pharmacokinetics of the plasmid-liposomal complexes were determined by injecting mice with complexes comprising the S ³⁵ -labeled DNA plasmid. 6 shows the percentage of injection as a function of time after intravenous injection. Immediately after injection of the plasmid-liposomal complex, an injection of about 7% is present in the bloodstream. In other studies, the remaining percentage of the complex was determined to be concentrated in the lung immediately after injection. After a period of time, the complex is neutralized by serum proteins in the lungs and enters the bloodstream, as evidenced by an increase in the percentage of injection to about 22% at 5 hours (FIG. 6). The complex then disappears from the bloodstream and there is an injection of about 12% after 24 hours.

Plasmid-complexes were prepared for in vivo administration with varying ratios of liposome lipids to plasmids. This complex was prepared according to the general procedure described in Example 1 while varying the amount of polycationic condensing agent and the total amount of liposome lipids. Typically, the amount of polycationic condensing agent was 100-500 μg and the ratio of liposome lipid / plasmid was μg of lipid / plasmid of 8-18 nmoles.

7-9 show the results for plasmid-liposomal complexes prepared with whole histones (FIGS. 7A-7E), histone H1 (FIGS. 8A-8B) and histone H4 (FIGS. 9A-9E) as polycationic condensing agents. After administration of the complexes prepared from the formulations shown in the table below, luciferase expression was measured in lung, liver, heart, spleen and kidney.

Table 1 shows the composition for plasmid-liposomal complexes prepared using all histones as polycationic condensing agents. The amount of total histone was varied between 100-500 μg to change the plasmid / total histone ratio to 0.2-1.0. The ratio of liposome lipid / plasmid was also varied and the ratio of μg of liposome lipid / plasmid of 8, 14 and 18 nmoles was tested.

ingredient Formulation number ¹ One 2 3 4 5 6  pNSL plasmid (μg) 100 100 100 100 100 100  Total histone (μg) 200 100 200 350 500 200 Liposome lipid ² (μmole) 0.8 1.4 1.4 1.4 1.4 1.8  Plasmid (μg) / Total histone (μg) 0.5 1.0 0.5 0.3 0.2 0.5  nmole lipid / plasmid (μg) 8 14 14 14 14 18  1: In Vivo Results for Each Formulation Number Illustrated in FIGS. 7A-7E 2: Liposomes Prepared from a 1: 1 Molar Ratio of DDAB: Cholesterol

Results of in vivo mouse administration of the formulations summarized in Table 1 are shown in FIGS. 7A-7E, where lungs (FIG. 7A), liver (FIG. 7B), heart (FIG. 7C), spleen (FIG. 7D), and Luciferase expression in kidney (FIG. 7E) is expressed in Relative Light Units (RLU) / 10 sec / mg protein. The figure shows that transfection has the highest range. In particular, for total histones as condensers, transfection is highest when the liposome lipid / plasmid ratio is greater than 8 μg of lipid / plasmid and less than 18 nmole of lipid / plasmid.

Table 2 summarizes the plasmid-liposomal complex compositions prepared and tested in vivo using histone H1 as the polycationic condensing agent. The ratio of plasmid / histone H1 is 0.3 or 0.5 and the liposome lipid / plasmid ratio varies at μg of lipid / plasmid of 8, 14 and 18 nmoles.

ingredient Formulation number ¹ 7 8 9 10 11  pNSL plasmid (μg) 100 100 100 100 100  Histone H1 (μg) 350 200 350 200 350 Liposome lipid ² (μmoles) 0.8 1.4 1.4 1.4 1.4  Plasmid (μg) / Histone H1 (μg) 0.3 0.5 0.3 0.5 0.3  Lipids / plasmid (μg) 8 14 14 18 18  1: In Vivo Results for Each Formulation Number Illustrated in FIGS. 8A-8E 2: Liposomes Prepared from DDAB: Cholesterol in a Molar Ratio of 1: 1

The results of in vivo administration of the formulations summarized in Table 2 to mice are illustrated in FIGS. 8A-8E, where lung (FIG. 8A), liver (FIG. 8B), heart (FIG. 8C), spleen (FIG. 8D) and kidneys. Luciferase expression in (FIG. 8E) is expressed in relative light units (RLU) / 10 sec / mg protein. The figure shows that the best transfection is achieved when the ratio of liposome lipid / plasmid is 14 and 18.

Tables 3a and 3b summarize the plasmid-liposomal complex formulations formed using histone H4 as the polyvalent cationic condensing agent. The ratio of plasmid / histone H4 was changed to 0.2, 0.3 or 0.5 and the liposome lipid / plasmid ratio was changed to μg of lipid / plasmid of 8, 14 or 18 nmoles.

ingredient Formulation number ¹ 12 13 14 15  pNSL plasmid (μg) 100 100 100 100  Histone H4 (μg) 200 350 500 200 Liposome lipid ² (μmoles) 0.8 0.8 0.8 1.4  Plasmid (μg) / Histone H4 (μg) 0.2 0.3 0.2 0.5  Lipids / plasmid (μg) 8 8 8 14  1: In vivo results for each formulation number illustrated in FIGS. 9A-9E: liposomes prepared from DDAB: cholesterol in a molar ratio of 2: 1: 1

ingredient Composition number 1 16 17 18 19 20  pNSL plasmid (μg) 100 100 100 100 100  Histone H4 (μg) 350 500 200 350 500 Liposome lipid ² (μmoles) 1.4 1.4 1.8 1.8 1.8  Plasmid (μg) / Histone H4 (μg) 0.3 0.2 0.5 0.3 0.2  Lipids / plasmid (μg) 14 14 18 18 18  1: In vivo results for each formulation number illustrated in FIGS. 9A-9E: liposomes prepared from DDAB: cholesterol in a molar ratio of 2: 1: 1

In vivo administration of the formulations summarized in Tables 3A and 3B to mice is illustrated in FIGS. 9A-9E, where lungs (FIG. 9A), liver (FIG. 9B), heart (FIG. 9C), spleen (FIG. 9D) and Luciferase expression levels in kidney (FIG. 9E) are expressed in relative light units (RLU) / 10 sec / mg protein. The largest range of transfection is present at greater than 8 μg of liposome lipids / plasmids to less than 8 nmoles of liposome lipids / plasmids.

In other experiments conducted to support the present invention, plasmid-liposomes using polyl-glutamine, melittin (low molecular weight peptides containing 26 amino acids) or polymyxin B sulfate as polycationic condensing agents The complex was prepared. These are each commercially available from Sigma Chemical Co. In vivo transfection was measured by injecting the complex into mice as described in Example 2 and measuring the amount of luciferase expression.

Table 4 summarizes the composition of plasmid-liposomal complexes prepared using poly-glutamine, melittin or polymyxin B as polycationic condensing agents. The amount of condensing agent was varied from 50 to 200 μg.

 Condensing agents Condensing Agents ¹ Poly-1-glutamine Melittin Polymyxin B  Formulation number 21 22 23 24 25 26 27 28 29  pNSL plasmid (μg) 100 100 100 100 100 100 100 100 100  Condensing Agent (μg) 50 100 200 50 100 200 50 100 200 Liposome lipid ² (μmoles) 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4  Plasmid (μg) / Condensing Agent (μg) 2 One 0.5 2 One 0.5 2 One 0.5  Lipids / plasmid (μg) 14 14 14 14 14 14 14 14 14  1: In Vivo Results for Each Formulation Number Illustrated in FIGS. 10A-10E 2: Liposomes prepared from DDAB: cholesterol in a molar ratio of 2: 1: 1

The results of in vivo administration of the plasmid-liposomal complexes of Table 4 to mice are illustrated in FIGS. 10A-10E, where lung (FIG. 10A), liver (FIG. 10B), heart (FIG. 10C), spleen (FIG. 10D) and kidneys. Luciferase expression in (FIG. 10E) is expressed in pg of luciferase / mg protein. The lipid / plasmid ratio in each of the formulations was constant at μg of lipid / plasmid of 14 nmoles, which ratio was about midpoint of the preferred range of μg of liposome lipid / plasmid of 5-25 nmoles.

According to the above studies using various polycationic condensing agents, for example whole histone, histone H1, histone H4, poly-glutamine, melittin and polymyxin B, transfection results in a liposome lipid / plasmid ratio (unit: was achieved when the μg of lipid / plasmid of nmoles was greater than 5 to less than about 25. More preferably, the ratio is μg of liposome lipid / plasmid of 8-18, even more preferably 10-15, most preferably 12-14 nmoles.

As discussed above, the plasmid / liposomal complexes of the present invention are stable, as evidenced by little change in particle size for 90 days at 4 ° C. (see FIG. 5). Expression stability of this complex was measured by administering to the mice plasmid-liposomal complexes stored at 4 ° C. Specifically, intravenous administration of plasmid-liposomal complex suspensions to mice immediately (0 days) after preparation of the complex and 7, 14, 21, 28, 30 and 90 days after storage at 4 ° C. It was. According to the method described in detail in Example 2, luciferase expression in lungs and liver was measured, and the results are shown in FIGS. 11A-11B.

As can be seen in FIGS. 11A-11B, the amount of luciferase expression as a function of the storage time of the complex in the lung (FIG. 11A) and liver (FIG. 11B) remained constant.

It was evident that the complex retained the ability to express the coding gene during the 90 day test period. Thus, in one embodiment of the present invention, the complex is characterized by the ability to maintain at least 50% of the expression activity measured at 0 days for at least 30 days, more preferably 90 days.

Dose-response studies were performed using the plasmid-liposomal complexes prepared as described in Example 1. Plasmid-liposomal complexes were administered intravenously to mice at five plasmid dose levels of 25, 50, 200, 250 and 200 μg. After 24 hours of administration, luciferase expression levels in lung, heart, liver, kidney and spleen were measured, and the results are shown in FIG. 12. Luciferase expression measured was proportional to dose, with the highest expression in lung.

The amount of systemic luciferase expression 24 hours after administration of the plasmid-liposomal complex in mice is shown in FIG. 13. Plasmid-liposomal complexes are widely distributed, as evidenced by luciferase expression in bone marrow, lymph nodes, and brain.

As described above in Example 3, in another study conducted to support the present invention, mice bearing metastatic Lewis lung tumors were treated with plasmid-liposomal complexes. After tumor inoculation, mice were treated with one of the eight treatments shown in Table 5 of Example 3. Treatment is administration of plasmid-liposomal complexes prepared using plasmids carrying genes encoding p53 (pCMVp53) and interleukin 2 (pCMVIL2), and plasmids prepared with pHSVtk (herpes simplex virus thymidine kinase) plasmid Combined treatment of liposome complexes with ganciclovir was included. Saline was administered to the control animal group, and the control group was administered a pNSL plasmid encoding luciferase (Example 1) or a complex prepared only with gancyclovir.

14A-14C show the percentage of surviving animals as a function of days after tumor-cell inoculation. In all of FIGS. 14A-14C, animals treated with saline are denoted by s. As illustrated, all saline-treated animals died 24 days after tumor inoculation. In FIG. 14A, a complex comprising 50 μg (▼) and 75 μg (□) pHSVtk plasmids, and an animal treated with gancyclovir are illustrated. All animals treated with gancyclovir (O) alone died at 37 days. In contrast, animals treated with the combination therapy of liposome-plasmid complex and gancyclovir were well and 70% of animals treated with high plasmid dose survived the study.

14B shows survival of animals treated with plasmid-liposomal complexes containing pCMVp53 (▼) and pNSL (luciferase coding) (O). As illustrated, 20% of animals treated with the complex comprising the plasmid encoding the p53 gene survived the study, whereas with the pNSL plasmid encoding the saline treated animals (■) and luciferase-reporter genes. The treated animals died.

FIG. 14C shows the results in animals treated with a complex comprising a pCMVIL2 plasmid administered 50 μg of plasmid DNA (▼) and 75 μg of plasmid DNA (□). As illustrated, between 15 and 20% of animals survived to the end of the treatment period, while all remaining untreated (saline control, ■) animals died on day 24. Similarly, animals (O) treated with complexes comprising luciferase encoding pNSL plasmids died on day 31.

Formulation No. 34 of Table 5 of Example 3, i.e., a plasmid-liposomal complex comprising the pNSL luciferase-encoding gene, was prepared in 50 μg of plasmid / 0.7 μmol of lipid, 75 μg of plasmid / 1.05 μmol of lipid and 100 Mice were dosed at a dose of μg plasmid / 1.4 μmol lipid to monitor the body weight of the mice as an indicator of toxicity of the formulation. As can be seen in FIG. 15, mice treated with complexes at 1, 8, 15, 22 and 28 days lost some weight after the first dose as indicated by arrows in the figure, but lost weight within a few days. Recovered. Symbols in the figures are as follows: plasmid-liposomes at doses of saline-treated mice (+ symbols), and 50 μg of plasmid (O), 75 μg of plasmid (▼), and 100 μg of plasmid (▽). Mice Treated with Complex.

In another study using the Lewis lung tumor model, pNSL-luciferase-plasmid-liposomal complexes were administered to mice, and luciferase expression levels in various tissues were examined 24 hours after administration of the plasmid-liposomal complex. As can be seen in FIG. 16, the greatest luciferase expression was observed in lungs and tumors.

From the above, it can be seen how various features and objects of the present invention are met. Plasmid-liposomal complexes prepared according to the methods of the present invention form a substantially uniform population having a size of less than about 200 nm, as evidenced by dynamic light scattering. The complex is stable for 90 days without aggregation of the complex as evidenced by the dynamic light scattering method. It is also important that the transfection activity of the complex is stable, which maintains at least 50% transfection efficiency after storage at 4 ° C. for at least 30 days.

IV. Example

The following examples illustrate the preparation, characterization and use of the plasmid-liposomal complexes of the present invention. These embodiments are by no means intended to limit the scope of the invention.

Materials and methods

A. Geology

Dimethyldioctadecylammonium (DDAB) was purchased from Avanti Polar Lipids Inc (Birmingham, AL). Cholesterol greater than 99% purity was purchased from Nu-Chek (Elysian, MN).

B. Polycationic Condensing agents

All histones, histones H1 and histones H4 consisting of a mixture of histones including H1, H2, H3 and H4 were obtained from Boehringer Mannheim (Indianapolis, Ind.). Poly-glutamine, melittin and polymyxin B sulphate are described in Sigma Chemical Co. (St. Louis, Mo.).

C. Method: Dynamic Light Scattering

Size distribution measurements were obtained by dynamic light scattering (DLS) using a Coulter N4MD instrument operated according to the manufacturer's instructions. This result is expressed as the mean diameter (nm) and the standard deviation of the Gaussian distribution of the particles by relative volume.

Example  One

Plasmid Liposomes  Preparation of the complex

A. Preparation of pNSL Plasmids

The pNSL plasmid encoding luciferase was constructed with two commercial plasmids, the pGFP-N1 plasmid (Clontech, Palo Alto, Calif.) And pGL3-C (Promega Corporation, Madison, Wis.).

pGL3-C was digested with Xba l and blunt-ended ligation was performed using Klenow fragments of E. coli DNA polymerase. This was then cleaved with Hind III, followed by gel-purification of the 1689-bp fragment carrying the luciferase gene. The 4.7 kb fragment isolated from the agarose gel after digesting the pGFP-N1 plasmid with Sma I and Hind III was ligated with the luciferase fragment. JM109 Escherichia coli cells were transformed to select 20 colonies; About half of them showed the presence of inserts; Eight clones with inserts were cleaved with Nam HI and Xho I to further confirm the presence of the luciferase gene; Seven of these were positive.

B. Cationic Preparation of Liposomes

Cationic liposomes are mainly composed of CHCl ₃ It was prepared according to standard methods by dissolving 10 μmol DDAB and 10 μmol cholesterol in an organic solvent containing. This lipid was spun under reduced pressure and dried into a thin film. This lipid membrane was hydrated with the addition of distilled water to form a liposome suspension at a concentration of 20 μmole / ml. Liposomes were sized by ultrasonication or sequentially extruded through pore sizes of 0.4 μm, 0.2 μm, 0.1 μm and 0.05 μm of Nucleopore polycarbonate membranes to obtain liposomes of size less than about 200 nm (Nucleopore Pleasanton, CA).

C. Preparation of Condensed Plasmids

The DNA plasmid pNSL encoding luciferase prepared as described above was condensed according to the following procedure. 400 μl of plasmid (1 mg / ml in distilled water) was diluted with 310 μl of distilled water and mixed with 90 μl of 50% glucose. 100 μl of a polycationic condensate (total histone, histone H1, histone H4, poly-1-glutamine, melittin or polymyxin B) taken from the stock solution (1 mg / ml in distilled water) was slowly added to the plasmid solution with stirring. . This mixture was stirred for 10 minutes.

D. Preparation of the Complex

Plasmid-liposomal complexes having a μg liposome lipid / plasmid ratio of 14 nmole of lipid / plasmid were prepared by diluting 280 μl of suspended liposomes with 350 μl of distilled water and then adding 70 μl of 50% glucose. The suspension of condensed plasmid was slowly added to the dilute cationic liposome suspension with continuous stirring for 10 minutes.

Example  2

In vivo Transfection  step

In vivo transfection with plasmid-liposomal complexes was performed with BALB / c mice obtained from Simonsen (Gilroy, CA). Each plasmid-liposomal complex formulation was injected into three mice via the tail vein. The mice were killed 24 hours after injection to collect tissue (lung, liver, spleen, kidney, heart) and perfused with heparinized PBS (4C) under anesthesia.

At a temperature of 0-4 ° C., 0.75 ml of cell lysis reagent (Promega, Madison, Wis.) Was added to each tissue and the tissue was homogenized for 1 minute at 20,000 rpm. The supernatant was placed in a microcentrifuge tube and spun at 10,000 g for 5 minutes. Supernatants were harvested and analyzed for luciferase and protein. 20 μl of each sample was measured immediately with a luminometer (100 μl luciferin and ATP containing assay buffer, 10 second measurement). Relative light units were normalized to the amount of protein in the extract.

Example  3

In vivo Transfection  Tumor bearing mice

A. Preparation of Plasmid- Liposome Complexes

Plasmid-liposomal complexes were prepared according to the procedure of Example 1 using the following plasmids: pNSL encoding luciferase prepared as described in Example 1, and pCMVp53, pCMVIL2 and pHSVtK (all commercially available).

B. Tumor Inoculation

80 B6C3-F1 male mice were obtained from Taconic Farms (German Town, NY) and incubated for 3 days prior to the start of the experiment. These animals were housed in well-separated cages fed with instant sterile rodent food and acidified water (12:12 contrast cycle). These animals were randomly extracted into pre-tumor treatment groups according to body weight. These animals were randomly extracted into pre-inoculation treatment groups of tumors.

All animals were observed daily for general condition. These animals were weighed before inoculation of tumor cells and weighed twice per week. Any animal that was observed to be at least 15% less than the initial body weight or was found to be tired was euthanized and examined for the presence and size of metastatic sites in the lungs, liver and spleen.

Tumors were inoculated by taking Lewis Lewis tumors growing from other B6C3-F1 mice by surgery under sterile euthanasia. The tumor was chopped as finely as possible mechanically and briefly digested (37 ° C.) in an enzyme mixture of collagenase, protease and DNAse. After digestion and washing in medium (RPMI + 15% FCS), cells were counted with hemocytometer. The cells were spun down and resuspended in medium at 10 ⁶ cells / ml (10 ⁵ cells / 0.1 ml injection). Resuspended cells were sucked up with individual syringes (0.1 ml, with continuous mixing) and injected intravenously into the tail veins of each animal.

C. Treatment

Animals were treated with one of the eight curing methods described in Table 5 below from day 3 post inoculation with tumor cells. Ten animals in each group were treated five times at weekly intervals, except as indicated for Gancyclovir treated group No. 30 and animals administered Gancyclovir as part of the combination therapy (groups 32,33).

Formulation number  Furtherance 30 ^1,2,3,4  Brine, 0.1ml IV 31 ¹  Gancyclovir, 100 μg / kgIP (2 times per day for 6 days for 5 weeks) 32 ¹  Liposome / pHSVtk complex, 50 μg DNA / mouse + gancyclovir 100 μg / kg (twice daily for 6 days after each administration of plasmid / liposomal complex) 33 ¹  Liposome / pHSVtk complex, 75 μg DNA / mouse + gancyclovir 100 μg / kg (twice daily for 6 days after each administration of plasmid / liposomal complex) 34 ^2,3,4,5  Liposome / pNSL (luciferase encoding) complexes; various doses of 50,75,100 μg of plasmid 35 ²  Liposome / pCMVp53 complex, 75 μg DNA / mouse 36 ³  Liposome / pCMVIL2 complex, 50 μg DNA / mouse 37 ³  Liposome / pCMVIL2 complex, 75 μg DNA / mouse  1 Data shown in FIG. 14A 2 Data shown in FIG. 14B 3 Data shown in FIG. 14C 4 Data shown in FIG. 15 5 Data shown in FIG.

Twenty four hours after the last treatment, surviving animals were euthanized to collect and examine tissues and tumors. The percentage of surviving animals for each treatment group is shown in FIGS. 14A-14C. The toxicity of Formulation No. 34 is shown in FIG. 15 and the luciferase expression level of Formulation No. 34 is shown in FIG. 16.

Although the present invention has been described in connection with specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

The present invention provides a plasmid-liposomal composition for transfection of cells. The composition comprises plasmid molecules and cationic liposomes condensed with a polycationic condensing agent. The present invention also provides a method for preparing a plasmid-liposomal complex.

Claims (1)

(i) condensed plasmid molecules, and (ii) cationic vesicle forming lipids with diacyl chains, dimethyldioctadecylammonium (DDAB), N- [1- (2,3-ditetedecyloxy) propyl] -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N- [1- (2,3-dioleyloxy) propyl] -N, N-dimethyl-N-hydroxy ethylammonium bromide (DORIE ), Or a gene contained in a plasmid containing cationic liposomes containing N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA). A composition used to transfect, Wherein the plasmid-liposomal complex is obtained from the condensed plasmid molecule and the cationic liposome by the following steps: (a) selecting a polycation selected from the group consisting of histones, poly-glutamine, protamine, melittin and polymyxin B as a condensing agent for condensation of plasmid molecules, (b) selecting an aqueous medium containing the non-electrolyte solute with an ionic strength of less than the ionic strength of a 10 mM solution containing monovalent ionizable salts as the suspending medium of the condensed plasmid molecules and the cationic liposomes, (c) selecting a ratio of liposome lipids to plasmids of more than 5 μg of liposome lipids / plasmids to less than 25 μg of liposome lipids / plasmids, and (d) mixing the condensed plasmid molecules and cationic liposomes in the suspension medium to form a plasmid-liposomal complex having a uniform size of less than 200 nm.

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