JP2006028061A - Targeting nucleic acids to the liver - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a preparation for increasing the efficiency of targeting and uptake of a nucleic acid to the liver in vivo.
A preparation for liver delivery, comprising a polyion complex formed from a nucleic acid and a cationized pullulan derivative, wherein the cationized pullulan derivative has a cationization rate of 10-27%.
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Description

  The present invention relates to a water-soluble polymer preparation for targeting a nucleic acid to the liver in gene therapy.

  Gene therapy is a method for treating diseases by controlling the function of cells by artificially supplementing genes or artificially suppressing gene expression. Development of a vector capable of efficiently expressing a gene for use in such gene therapy is required. Gene expression vectors are roughly divided into cases where viruses are used and cases where viruses are not used. Until now, viruses such as adenoviruses, retroviruses, adeno-modified viruses have been mainly used as vectors because of their high expression efficiency. However, there are problems due to the original toxicity and immunogenicity of the virus, and molecular biological studies are under way to solve this problem. On the other hand, the latter method that does not use a virus includes the use of a non-viral gene carrier or physical stimulation such as electricity and ultrasound, but in either case, the gene transfer efficiency is extremely low. So far, non-viral gene carriers such as calcium phosphate, cationic polymers or liposomes have been researched and developed. A method of combining them with electrical stimulation has also been devised.

  Issues to be solved in the development of gene therapy include inhibiting gene degradation in vivo, increasing the efficiency of targeting to target organs, enabling stable sustained release in target organs, and cells Increasing the efficiency of gene transfer into the body. There are in vivo methods and in vitro methods for gene transfer. The extracorporeal method involves combining a gene with a vector and culturing it with a cell, and the key is how to successfully interact the gene with the cell. On the other hand, in the in vivo method, in addition to that, the stability of the gene in the body is increased, and how the gene can be targeted to a specific site greatly affects the expression at the gene level. For example, cationic gene carriers are likely to interact with biological components such as proteins, lipids, blood cells, etc., and it is extremely difficult to control their pharmacokinetics. Thus, attempts have been made to modify the pharmacokinetics of the gene and improve its stability by reducing the interaction with biological components with a molecule having high affinity for specific cells or tissues and polyethylene glycol.

  In order to increase the efficiency of gene targeting and intracellular introduction, a drug delivery system combining a gene with a polymer carrier has been proposed. One such polymer carrier is pullulan. Pullulan is a natural polysaccharide with a high affinity for the liver (Yamaoka et al., Drug Delivery, 1, 75-82 (1993)) and via the asialoglycoprotein receptor expressed in hepatocytes. Incorporation into hepatocytes (Kaneo et al., J. Control Release, 70, 365-373 (2001)) is known. Therefore, if pullulan having such characteristics is used as a gene DDS carrier, it is considered that the gene can be targeted to the liver. For example, Japanese Patent Application Laid-Open No. 2003-104914 discloses a complex for targeting to the liver obtained by binding plasmid DNA and pullulan-DTPA via a metal coordination bond.

Prior art document information related to the present invention includes the following.
JP2003-104914A Yamaoka et al., Drug Delivery, 1, 75-82 (1993) Kaneo et al., J. Control Release, 70, 365-373 (2001)

  An object of the present invention is to increase the efficiency of targeting a nucleic acid to a liver in vivo and the efficiency of intracellular uptake by binding a nucleic acid and a water-soluble polymer by ionic bonds.

  In the preparation for liver delivery using a polyion complex formed from a nucleic acid and a cationized pullulan derivative, the present inventors have adjusted the cationization rate of the cationized pullulan derivative to a specific range, thereby allowing the nucleic acid to enter the liver in vivo. I found that the efficiency of importing was increased.

  The present invention provides a preparation for liver delivery, comprising a polyion complex formed from a nucleic acid and a cationized pullulan derivative, wherein the cationized pullulan derivative has a cationization rate of 10-27%. Here, the cationization rate of the cationized pullulan derivative represents the introduction molar ratio of cations per hydroxyl group of pullulan. Preferably, the cationized pullulan derivative in the present invention has a cationization rate of 15-26%, more preferably 20-25%, still more preferably 22-24%, and most preferably 23-24%. is there.

  Pullulan is an α-glucan fermented and produced by Aureobasidium pullulans using a partial hydrolyzate of starch as a raw material, and is a linear water-soluble chain in which maltotriose consisting of three glucoses is linked by α-1,6 bonds. It is a polymer. Widely used as food additives and pharmaceutical supplements, products of various molecular weights are commercially available. It is known that pullulan has high affinity for the liver and is taken into hepatocytes via the asialoglycoprotein receptor expressed in hepatocytes. Preferably, the molecular weight of the pullulan used in the present invention is about 10,000 or more, more preferably about 20,000 or more, more preferably about 30,000 or more, and most preferably about 40,000 or more. Also preferably, the molecular weight of the pullulan used in the present invention is about 200,000 or less, more preferably about 120,000 or less, more preferably about 80,000 or less, and most preferably about 60,000 or less.

  In the present invention, a cationized pullulan derivative having a cationic group introduced into pullulan is used to form a polyion complex with a nucleic acid. The cationization step is not particularly limited as long as it is a method capable of introducing a functional group that can be cationized under physiological conditions. However, a mild condition in which a 1, 2 or tertiary amino group or an ammonium group is added to the hydroxyl group on the pullulan molecule. The method introduced below is preferred. For example, alkyldiamines such as ethylenediamine, N, N-dimethyl-1,3-diaminopropane, trimethylammonium acetohydrazide, spermine, spermidine, or diethylamide chloride may be used in combination with various condensing agents such as 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide hydrochloride, cyanuric chloride, N, N'-carbodiimidazole, cyanogen bromide, diepoxy compound, tosyl chloride, diethyltriamine-N, N, N ', N ", N" -pentane The reaction can be carried out using a dianhydride compound such as acid dianhydride, trisyl chloride and the like. In particular, the method of reacting ethylenediamine or spermine is convenient and versatile.

  The nucleic acid used in the present invention includes DNA, RNA, DNA-RNA complex, siRNA, PNA, and derivatives thereof having modifications in sugar, phosphate or base. Examples of nucleic acids include genes encoding proteins useful for the treatment of liver diseases, vectors containing the same, antisense DNA and decoy DNA (for example, NFκB, etc.) that can be used for liver diseases such as cancer Can be mentioned. Examples of genes encoding proteins necessary for the treatment of liver diseases include low molecular weight peptides such as hormones, or proteins such as interferons, interleukins, cytokines, chemokines, cell growth factors or matrix metalloproteases (MMPs), and the like. Examples thereof include partial peptides at the physiologically active site of proteins, or genes such as neutralizing antibodies and receptor agonists of these proteins and peptides. The low molecular weight peptide such as a hormone is not particularly limited as long as it is particularly suitable for treatment, and examples thereof include IFN and the like. Examples of cell growth factors include hGH, EGF, NGF, HGF, FGF, HB-EGF, IGF, and the like, and fragments thereof such as NK4, which is an intramolecular fragment of HGF.

  DNA encoding a therapeutically useful protein may be obtained by cloning from a genomic or cDNA library based on known sequences, or may be produced by chemical synthesis. The DNA encoding the protein is used after being introduced into a plasmid vector so that the function of the protein can be expressed in the introduced cell. The plasmid vector includes a promoter region, a start codon, a stop codon, a terminator region, and the like arranged in such a manner that DNA is transcribed in the cell and the protein encoded thereby is appropriately expressed. Such a plasmid vector can be easily prepared by inserting a desired DNA into an expression vector available in the art using an appropriate restriction enzyme site. It can also be prepared by synthetic or semi-synthetic means based on the base sequence of the DNA to be introduced. The type of promoter, start codon, stop codon, and terminator region in the plasmid vector are not particularly limited.

  As long as the protein expressed from the DNA in the preparation of the present invention has a desired therapeutic activity, one or more amino acids in the amino acid sequence may be substituted, deleted, and / or added. Similarly, sugar chains may be substituted, deleted and / or added. Furthermore, it may be produced as a fusion protein with another protein or a fragment thereof. Therefore, the DNA may encode such a modified protein. Methods for producing DNAs encoding such modified proteins by site-directed mutagenesis, gene recombination methods or synthesis methods are well known in the art.

  A polyion complex is a complex formed by binding a nucleic acid and a cationized pullulan derivative by an ionic bond. A polyion complex can be formed by mixing a nucleic acid and a cationized pullulan derivative in a suitable buffer solution and allowing to stand for a predetermined time. The reaction can be carried out at room temperature. Formation of the polyion complex can be measured using the turbidity of the mixed solution as an index. By forming a polyion complex of a nucleic acid and a cationized pullulan derivative according to the present invention, the negative charge of the nucleic acid is neutralized and the molecular size is reduced due to relaxation of the electrical repulsion. In addition, the nucleic acid is stabilized in the formulation. Furthermore, due to the high affinity between pullulan and hepatocytes, nucleic acids can be incorporated into hepatocytes with high efficiency.

  The polyion complex of the present invention is water-soluble, and can be used as it is in an assay or treatment by dissolving it in a diluent such as a buffer solution, physiological saline, or an injection solvent. Alternatively, it may be used after being lyophilized and then dissolved in a diluent at the time of use. Examples of the administration method include intravenous administration, intraarterial administration, intradermal administration, subcutaneous administration, intramuscular administration, and intracavitary administration. Particularly preferred is intravenous administration. Alternatively, it may be injected directly into the portal vein using a catheter.

  The dosage of the formulations of the present invention can be appropriately selected so that it is sufficient to produce a therapeutic response. The dose is usually selected from the range of about 0.001 to about 100 μg, preferably about 0.01 to about 10 μg of nucleic acid per adult patient. In addition, when the effect is insufficient with a single administration, the administration can be performed a plurality of times.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Preparation of Cationized Pullulan Derivative The preparation of the cationized pullulan derivative was performed by introducing N, N′-bis (3-aminopropyl) -1,4-butanediamine (spermine) into the hydroxyl group of pullulan. Pullulan (manufactured by Shodex) having a weight average molecular weight of 47,300 was dissolved in dehydrated dimethyl sulfoxide (10 mg / ml). Next, various concentrations of N, N′-carbonyldiimidazole (CDI) and spermine were added and stirred at room temperature for 20 hours. The reaction solution was dialyzed against distilled water for 2 days and freeze-dried to obtain a spermine-introduced cationized pullulan derivative.

  The cationization rate of the spermine-introduced cationized pullulan derivative was calculated by quantification of amino groups using 2,4,6-trinitrobenzenesulfonic acid (TNBS) method. That is, 100 μl of 0.2 M phosphate buffered saline (PBS, pH 7.4) 100 μl, 200 μl of 4 wt% aqueous sodium bicarbonate and 200 μl of 0.1 wt% TNBS aqueous solution were added to 100 μl of an aqueous solution of a spermine-introduced cationized pullulan derivative. And reacted at 37 ° C. for 2 hours. The absorbance at 415 nm of this reaction solution was measured. From this absorbance value and a calibration curve using β-alanine, the molar ratio of spermine introduced per hydroxyl group of pullulan was calculated, and this was defined as the cationization rate (%).

  Table 1 shows the production conditions and cationization rate of the produced cationized pullulan derivatives. By changing the concentrations of pullulan, spermine and CDI, cationized pullulan derivatives having various cationization rates could be obtained.

Formation of polyion complex of cationized pullulan derivative and plasmid DNA Plasmid DNA encoding luciferase was used as a plasmid DNA gene. The luciferase plasmid DNA containing the ampicillin resistance gene was transformed into Escherichia coli and then cultured in ampicillin-containing LB medium at 37 ° C. for 18 hours. The proliferated Escherichia coli was collected by centrifugation, and plasmid DNA was extracted by the alkali-SDS method. As a purity evaluation of plasmid DNA, the ratio of 260 nm to 280 nm of the obtained plasmid DNA aqueous solution was measured and found to be between 1.8 and 2.0.

  An aqueous plasmid DNA solution (25 μg / ml) and a cationized pullulan derivative having a different cationization rate were mixed in a 10 mM phosphate buffer solution (pH 7.4) containing 150 nM NaCl so that the N / P ratio was 5. The N / P ratio is the ratio between the number of moles of amino groups in the cationized pullulan derivative and the number of moles of phosphate groups in the nucleic acid. By allowing this to stand at room temperature for 15 minutes, both polyion complexes were formed.

Measurement of molecular size The molecular size of the obtained polyion complex was measured using dynamic light scattering (DLS). This aqueous solution was measured with a DLS apparatus (manufactured by Otsuka Electronics) equipped with an Ar laser (detection angles 90 °, 37 ° C.). The hydrodynamic radius R of the polyion complex is the scattering intensity-time correlation function obtained by measurement and the Einstein-Stokes equation: R = kT / 3πηD (k: Boltzmann constant, T: absolute temperature, η: solvent viscosity, D: Nucleic acid coefficient) and calculated with the attached analysis software. It was possible to change the cationization rate of the pullulan derivative by changing the amount of charged CDI. Table 2 shows the results of measurement of the apparent molecular size and zeta potential of polyion complexes of cationized pullulan derivatives (N / P = 5) and plasmid DNA having different cationization rates.

  In the case of pullulan having a low cationization rate, a molecular size larger than that of free plasmid DNA was observed, and when the cationization rate was high, a decrease in molecular size was observed, and the value was approximately 200 nm. The zeta potential was approximately 12 mV or more at a cationization rate of 20% or more.

Measurement of turbidity The turbidity of the obtained polyion complex aqueous solution was measured. The wavelength used was 500 nm, and the change in absorbance of the aqueous solution was measured as turbidity. The results are shown in FIG. The turbidity of the mixed aqueous solution of cationized pullulan derivative and plasmid DNA increases with increasing cationization rate, and the molecular size of the formed polyion complex decreases, indicating that it interacts more strongly with plasmid DNA. It was. Moreover, since turbidity decreased as the ionic strength increased, it was confirmed that the interaction between pullulan-spermine and plasmid DNA was an electrostatic interaction, and a polyion complex was formed.

Evaluation of interaction between intercalator and plasmid DNA Ethidium bromide (EtBr, 0.4 μg / ml) was added to a 20 μg / ml plasmid DNA aqueous solution to intercalate EtBr into DNA molecules. An aqueous cationized pullulan derivative solution was added to this aqueous solution. The fluorescence intensity (excitation wavelength: 510 nm, fluorescence wavelength: 590 nm) before and after mixing of the cationized pullulan derivative aqueous solution was measured, and the relative fluorescence intensity was calculated with the fluorescence intensity of the original plasmid DNA / EtBr as 100%. In general, fluorescence is generated by Etcal intercalating into DNA. When the plasmid DNA and the cationized pullulan derivative form a polyion complex, EtBr cannot be intercalated with the plasmid DNA and is released from the plasmid DNA. That is, a decrease in relative fluorescence intensity indicates that an interaction between the cationized pullulan derivative and the plasmid DNA has occurred. The strength of the decrease is thought to correspond to the strength of interaction with the plasmid DNA cationized pullulan derivative.

  The results are shown in FIG. It was found that pullulan-spermine and plasmid DNA strongly interacted with pullulan having a high cationization rate because the fluorescence intensity decreased more rapidly than that with low pullulan. In addition, since the fluorescence was rapidly quenched when the N / P ratio increased, it was also found that pullulan-spermine and plasmid DNA interacted strongly.

Human liver cancer-derived strain HepG2 cells were used as experimental cells for in vitro gene transfer into cells. A 6-well plate (Costar) was seeded with 4 × 10 ⁴ cells / cm ² (4 × 10 ⁵ cells / well), and MEM (MEM-FCS) supplemented with 10 vol% fetal calf serum (FCS) was added. ) And cultured at 37 ° C. for 24 hours at 5% CO ₂ /95% air. After changing to Opti-MEM (Gibco) medium without serum, a polyion complex of luciferase-plasmid DNA and a cationized pullulan derivative (plasmid DNA amount 5 μg) or luciferase-plasmid DNA as a control was added for 6 hours. Cultured. Next, the medium was replaced with MEM-FCS, and the culture was further continued for 42 hours. After removing the medium and thoroughly washing the cells with PBS, cell lysate (25 mM Tris-phosphate buffer (pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N, N, N ′ , N′-tetraacetic acid, 10% glycerol, 1% Triton® X-100) was added to lyse the cells. Gene expression was quantified by measuring the chemiluminescence of luciferase protein in the cell lysate. Further, the total amount of protein in the cell lysate was measured by Bicinchonate (BCA) method. ANOVA was used for statistical analysis. A significant difference was assumed when the p-value was lower than 0.05.

  FIG. 3 shows in vitro gene transfer results into HepG2 cells using a polyion complex consisting of plasmid DNA and cationized pullulan derivatives having various cationization rates. As the cationization rate of the pullulan derivative increased, the gene expression level in HepG2 cells by the polyion complex increased. This is thought to be because the uptake and stability of plasmid DNA increased by forming a complex with a pullulan derivative having a high cationization rate. Further, since the gene expression level was significantly decreased in the presence of asialofetin (data not shown), it was found that the polyion complex was taken into the cell via the receptor.

In vivo gene transfer experiment into the liver 100 μl of polyion complex consisting of plasmid DNA and cationized pullulan derivatives with various cationization rates (injection amount of plasmid DNA: 10 μg) was injected into the tail vein of mice. After 24 hours, the mice were sacrificed by washing out the blood and the liver was removed. The liver was weighed and then homogenized at 4 ° C. using a Polytron homogenizer to prepare a tissue lysate. After centrifugation at 4 ° C. and 14000 rpm for 10 minutes, the supernatant obtained was the same as in Example 3. Then, luciferase activity was measured.

  The results are shown in FIG. In gene transfer in vivo, high gene expression was observed when a pullulan derivative having a specific cationization rate was used. This indicates that the intracellular uptake and stability of plasmid DNA increases with increasing cationization rate. Conversely, as the cationization rate decreases, the zeta potential of the polyion complex decreases, and as a result, the balance between the two properties of non-specific interaction with biological components decreases. It shows that the phenomenon is affected.

FIG. 1 shows the results of turbidity measurement of a polyion complex. FIG. 2 shows changes in the fluorescence intensity of the plasmid DNA-EtBr complex by mixing with a cationized pullulan derivative. FIG. 3 shows the results of in vitro gene transfer into HepG2 cells using a polyion complex. FIG. 4 shows the results of in vivo gene transfer into the liver using a polyion complex.

Claims (1)

A preparation for liver delivery, comprising a polyion complex formed from a nucleic acid and a cationized pullulan derivative, wherein the cationized pullulan derivative has a cationization rate of 10 to 27%.

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