JP5132958B2 - Hydrogel ophthalmic lens used for gene therapy - Google Patents

Description

  The present invention relates to a hydrogel ophthalmic lens that can be used for gene therapy. More specifically, the present invention relates to a hydrogel ophthalmic lens having a phosphate group capable of effectively introducing a gene into cells of ocular tissue.

  Genetic diseases are known to be caused by mutations in genes expressed in cells. As a treatment for such a genetic disease, a method of introducing a normal gene into an abnormal cell is promising.

  Conventionally, introduction of a gene into a cell has been performed by appropriately selecting a calcium phosphate method, an electroporation method, a method using a liposome, or the like. Among them, the calcium phosphate method has an advantage that a special instrument is not required, but has a disadvantage that gene transfer efficiency is low. Due to this drawback, the calcium phosphate method is used as a gene transfer method into cultured cells, but is not used as a gene transfer method into cells in vivo.

  Recently, as an attempt to introduce a gene directly into the living body, a liposome containing the gene is prepared and administered by injection or a biodegradable polymer carrying the gene is introduced directly into a living tissue by a surgical technique. The technique to take is taken.

  As an example using a liposome, there is an eye drop containing a liposome encapsulating an expression vector incorporating a gene DNA that can be expressed in mammalian cells (see Patent Document 1). This is a method of introducing a gene into an eye cell by eye drops for the purpose of gene therapy for an abnormal gene DNA involved in an eye disease caused by a gene abnormality. However, since the gene is diluted with tears immediately after instillation, it is necessary to devise a method for making the contact time with the cornea as long as possible.

  Furthermore, there is a method in which a gene is supported on a biodegradable polymer, and the gene is gradually released along with the collapse of the polymer so that high gene transfer efficiency can be obtained (see Patent Documents 2, 3, and 4). For example, there is a cationic biodegradable polymer that can efficiently carry an anionic gene. This is because cationic gelatin is loaded with an anionic gene by ionic interaction and implanted into the living body, and the gene is continuously released along with the degradation of gelatin, thereby efficiently introducing the gene into the cell. It is a technology that can be done. However, in this method, the gene introduced into the cell remains in the living body as a complex with degraded cationic gelatin, and the gene is introduced depending on the safety of the degradation product and the size of the gelatin-gene complex. There are concerns about problems such as changes in efficiency.

In addition, there is a method in which granules of calcium phosphate are incorporated into a hydrogel such as collagen and the gene is introduced into the cells using this calcium phosphate (see Patent Document 5). In this method, if a hydrogel containing calcium phosphate granules is processed into a desired shape such as a pellet, it can be directly inserted into a living body, and can be administered by injection because it is in a gel form. . However, it is indispensable to maintain high transparency of the cornea among the eye tissues targeted by the present invention. However, it is known that the cornea becomes clouded by the deposition of calcium. The method cannot be used.
Japanese Patent Laid-Open No. 10-67688 JP 2001-199903 A JP 2003-286200 A JP 2003-169844 A JP 2004-283138 A

  An object of the present invention is to provide a hydrogel ophthalmic lens capable of easily performing gene therapy for an abnormal gene involved in a disease caused by a gene abnormality. More specifically, an object of the present invention is to provide a hydrogel ophthalmic lens for practical gene therapy that can carry a gene that can be expressed in a cell and can exhibit a good sustained release effect.

  The present invention is a hydrogel ophthalmic lens comprising a gene carried after a treatment in which a hydrogel having a phosphate group is immersed in an aqueous solution containing a divalent metal ion. .

  Further, the present invention is the above-mentioned hydrogel ophthalmic lens, wherein the hydrogel having a phosphate group is a gel comprising at least a monomer having a phosphate group and a nonionic hydrophilic monomer. .

  The present invention is also the above-described hydrogel ophthalmic lens, wherein the divalent metal ion is a calcium ion, and the gene is a plasmid DNA.

  The hydrogel ophthalmic lens of the present invention releases a gene that can be expressed in the cells of the eye tissue without adversely affecting the cells of the eye tissue, particularly the cornea, over 24 hours, and efficiently into the cells of the eye tissue. It is practical as a gene release device for gene therapy that can introduce a gene.

  The present inventor, as a means for introducing a gene used for gene therapy into cells of eye tissue, a method of effectively releasing the gene with a hydrogel ophthalmic lens having a phosphate group, in particular, an anionic property This is based on knowledge obtained by earnestly examining a method using a complex in which a phosphate group in a gene and a hydrogel is formed through a divalent metal ion typified by calcium ion.

  That is, the present invention treats a hydrogel ophthalmic lens having a phosphate group in the presence of a divalent metal ion in order to effectively use a gene used for gene therapy, thereby efficiently producing the gene. It carries and discharges. Specifically, an appropriate interaction is exerted between the anionic gene and the hydrogel to effectively carry and gradually release the gene.

  Examples of the gene used for gene therapy preferably used in the present invention include plasmid DNA containing at least one anionic functional group in the molecular structure. Plasmid DNA is circular DNA and can be used as it is, but a desired gene can be conjugated in accordance with the intended treatment. For example, colloidal corneal dystrophy is an autosomal recessive genetic disease that causes severe amyloid deposition in the corneal epithelium. In this disease, amyloid is deposited on the cornea, so that transparency is lost and visual acuity is reduced. So far, it has been found that this causative gene is M1S1 (membrane component, chromosome 1, surface marker 1) gene. This M1S1 gene can be incorporated into plasmid DNA to serve as a carrier and introduced into the target eye cell. For example, when the hydrogel ophthalmic lens of the present invention, which has been treated in the presence of calcium ions, is immersed in an aqueous solution in which the target gene, particularly plasmid DNA, is dissolved, the plasmid DNA can be included in the lens. .

  The gene concentration in the aqueous solution should be appropriately selected for each gene depending on its solubility and the minimum effective concentration and maximum safe concentration for introduction into the target cell. A concentration of 0.1 μg / μL is preferred.

  In the present invention, a phosphoric acid group in a hydrogel and a gene form a complex through a divalent metal ion, so that the gene is continuously released to cells of ocular tissue. The monomer having a phosphoric acid group is preferably a compound represented by the following structural formula (I) and / or (II), and is preferably a compound represented by the formula (I). Particular preference is given to some methacryloxyethyl phosphate (MOEP; n = 2). In addition, the hydrogel ophthalmic lens of the present invention can change the amount of the gene carried according to the content of the monomer having a phosphate group, and can be appropriately selected according to the target cell. The amount of the monomer having a phosphate group is preferably 50% by weight or less of the monomer having a phosphate group with respect to the nonionic hydrophilic monomer to be used. If the content of the monomer having a phosphoric acid group exceeds 50% by weight, the gel becomes brittle, which is not practical.

  Any nonionic hydrophilic monomer may be used as long as it has at least one hydrophilic group in the molecule and is nonionic. For example, 2-hydroxymethyl (meth) acrylate, (meth) acrylamide, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, 2,3-dihydroxypropyl (meth) acrylate and 2-polyethylene glycol mono (Meth) acrylate, 2-polypropylene glycol (meth) acrylate, N-vinylpyrrolidone and the like can be mentioned, and two or more of these can be used in combination. The blending amount of the hydrophilic monomer is determined by the relationship with the blending amount of the phosphate group, but is preferably in the range of 50 to 95% by weight based on the total monomers. If these hydrophilic monomers are also appropriately selected, the water content of the hydrogel ophthalmic lens can be changed.

  In the present invention, in addition to the above components, any copolymerizable monomer can be used. For example, a crosslinkable monomer may be used when forming a network structure of a hydrogel ophthalmic lens and adjusting the mechanical strength. Examples of the crosslinkable monomer include ethylene glycol dimethacrylate, methylene bisacrylamide, 2-hydroxy-1,3-dimethacryloxypropane, trimethylolpropane triacrylate, and the like. The amount of the crosslinkable monomer used is preferably 0.1 to 4.0% by weight based on 100% by weight of the main monomer. Particularly preferred is 0.1 to 1.0% by weight. When the crosslinkable monomer is less than 0.1% by weight, there is no significant effect on the shape stability of the hydrogel ophthalmic lens, and when it exceeds 4.0% by weight, the crosslinking effect becomes excessive and the hydrogel eye The lens for use becomes fragile, which is not preferable.

  As other copolymerization components, hydrophobic monomers do not impair the effects of the present invention in order to adjust the water content and swelling rate of hydrogel ophthalmic lenses and to finely adjust the amount of the gene to be loaded. Can be used in a range. Any hydrophobic monomer may be used as long as it is compatible with the hydrophilic monomer and the monomer having a phosphate group, which are essential components of the present invention. For example, methyl methacrylate, isobutyl methacrylate, 2, 2, 2 -Trifluoromethacrylate, cyclohexyl methacrylate and the like can be mentioned.

  A feature of the present invention is that a gene is carried after a treatment in which a hydrogel having a phosphate group is immersed in an aqueous solution containing a divalent metal ion. The purpose of this is to efficiently carry a gene in the hydrogel by forming a complex via a divalent metal ion between the phosphate group and the gene in the hydrogel ophthalmic lens. The divalent metal ion in the present invention can be used as long as it is a metal ion that is highly safe for a living body, but is an alkaline earth metal ion or an amphoteric metal ion, such as magnesium, iron, copper, zinc, and the like. Of these, calcium ions are more preferable. For example, when calcium ions are used, a gene composed of a phosphate group-calcium-gene via this calcium ion can be formed by carrying the gene on a hydrogel ophthalmic lens to which the calcium ion is bound. it can. A complex composed of this moderate binding force is formed in the hydrogel ophthalmic lens, and the hydrogel ophthalmic lens is brought into contact with cells of the eye tissue to replace ions in the living body. Alternatively, the gene can be introduced into the cell in a gene-calcium-gene binding mode. By this method, the gene can be introduced in the same form as the calcium phosphate method, which is a conventional method for gene introduction into cells, and since the ocular lens shape increases the contact time with the eye cells, Introduction efficiency is also improved.

  The divalent metal ion concentration of the aqueous solution for treating the hydrogel ophthalmic lens can be appropriately selected according to the phosphate group content in the hydrogel, but is preferably in the range of 3 to 100 mM. More preferably, it is about 10 mM. Even if the hydrogel ophthalmic lens is treated with an aqueous solution of less than 3 mM, the formation efficiency of the complex consisting of phosphate group-metal ion-gene is lowered, and the amount of gene carried in the hydrogel ophthalmic lens is also lowered. . Further, when the treatment is performed at a concentration exceeding 100 mM, calcium may aggregate, crystallize, and precipitate on the surface of the hydrogel ophthalmic lens. In addition, the treatment may be performed by immersing the hydrogel ophthalmic lens in an aqueous solution containing divalent metal ions at room temperature for 1 hour or more, and then immersing in the aqueous gene solution. The extent which does not exceed is preferable. Further, the treatment time can be appropriately selected as long as it is 1 hour or longer. However, in the case of treatment for 3 hours or longer, the metal ions in the hydrogel become excessive, and therefore it is preferable that the treatment time does not exceed 3 hours.

  In the production of the hydrogel ophthalmic lens of the present invention, a polymerization initiator is first added to the monomer mixture, and further stirred and dissolved. As the polymerization initiator, any radical polymerization initiator can be used, and the addition amount of the polymerization initiator is preferably about 10 to 3500 ppm external to the total amount of monomers. The polymerization is carried out by putting the monomer mixture into a desired mold such as metal, glass or plastic and sealing it.

  After completion of the above polymerization, it is cooled to room temperature, and the resulting polymer is removed from the mold and cut and polished as necessary. The obtained polymer is hydrated and swollen to obtain a hydrogel ophthalmic lens. Examples of the liquid (swelling liquid) used for this hydration swelling include water, physiological saline, isotonic buffer and the like. The swelling liquid is heated to 60 to 100 ° C. and dipped for a certain period of time to quickly enter a hydrated and swollen state. Moreover, it becomes possible to remove the unreactive monomer contained in the polymer by the swelling treatment.

  Next, after being immersed in a buffer adjusted to pH 8 to 10 at room temperature for about 1 hour and subjected to alkali treatment, the hydrogel ophthalmic lens was sufficiently washed with pure water and adjusted in advance 2 It is immersed in an aqueous solution containing a valent metal ion, preferably calcium ion. After the treatment, the hydrogel ophthalmic lens is sufficiently washed with pure water, and the hydrogel ophthalmic lens of the present invention is obtained by immersing it in an aqueous solution in which a gene prepared in advance is dissolved.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples. In this example, plasmid DNA was used as a gene.

(Evaluation method)
<Evaluation of shape stability>
The ophthalmic lens before and after plasmid DNA release was evaluated in saline using a contact lens projector. For the ophthalmic lens before the release of the plasmid DNA, “◯” was given when no change in shape was observed, and “X” was given when deformation was observed.

<Measurement of light transmittance>
The light transmittance of the lens after carrying plasmid DNA was measured using an ultraviolet-visible spectrophotometer (UV-3150: Shimadzu Corporation).

<Measurement of moisture content>
The water content of the lens after carrying the plasmid DNA was measured according to “Measurement of water content of hydrogel lens (ISO10339: 1997)”.

<Measurement of gene release>
The lens after carrying the plasmid DNA was immersed in 3 mL of physiological saline at 25 ° C., and the amount released was measured by electrophoresis with an agarose gel every hour. 1 kbp was used as a standard marker for measurement. When the release of plasmid DNA was observed over 24 hours, it was marked with ◯, and when it was not recognized, it was marked with ×.

<Evaluation of release into tears>
The lens after carrying the plasmid DNA was attached to a rabbit, and 5 μL of tear fluid was collected with a capillary tube every hour. The collected plasmid DNA in tears was amplified by PCR (Polymerase Chain Reaction) method, and the presence of plasmid DNA in tears was confirmed by electrophoresis using agarose gel. For electrophoresis, 1 kbp was used as a standard marker. When the presence of plasmid DNA was observed in the tears for 12 hours, it was marked as ◯, and when it was not recognized, it was marked as x.

<Evaluation of introduction of rabbit corneal epithelial cells>
The lens after carrying the plasmid DNA was attached to a rabbit, and the rabbit corneal epithelial cells after the attachment were collected, cultured for 48 hours, and observed with a fluorescence microscope. The cells into which the plasmid DNA was introduced were colored with fluorescence by GFP (Green Fluorescent Protein) to evaluate the introduction of the plasmid DNA into the corneal epithelial cells. When introduction into corneal epithelial cells was observed, it was marked as ◯, and when not introduced, it was marked as x.

Example 1
Add 0.3% by weight of azobisisobutyronitrile (AIBN) (external) to 10% by weight of methacryloxyethyl phosphate (MOEP; n = 2) and 90% by weight of 2-hydroxyethyl methacrylate (HEMA). The mixture was stirred for about 1 hour while sufficiently purging with nitrogen. After stirring, the monomer mixture was placed in a plastic mold for an ophthalmic lens and polymerized by raising the temperature in the range of 50 to 100 ° C. over 25 hours. The polymer was taken out of the container, immersed in distilled water at about 80 ° C. for about 4 hours, and hydrated and swollen to obtain an ophthalmic lens of this example. This ophthalmic lens was immersed in a phosphate buffer solution (PBS) adjusted to pH 8 at room temperature for 1 hour for alkali treatment, and then thoroughly washed with pure water. The washed ophthalmic lens is immersed in a 10 mM aqueous solution of calcium chloride at room temperature for 2 hours, then washed with pure water, and immersed in 3 mL of a 0.1 μg / μL aqueous solution of plasmid DNA (CLONTEC) at 25 ° C. for 48 hours. To carry plasmid DNA. The ophthalmic lens carrying the plasmid DNA was immersed in 3 mL of distilled water at 25 ° C. for 24 hours to remove the free plasmid DNA, and then subjected to the various evaluations described above.

(Examples 2 to 8)
An ophthalmic lens having the composition shown in Table 1 and hydrated and swollen in the same manner as in Example 1 was obtained. This ophthalmic lens was treated with an aqueous calcium chloride solution adjusted to the calcium ion concentration shown in Table 1, and plasmid DNA was supported in the same manner as in Example 1 to perform the above various evaluations.

(Comparative Examples 1-2)
An ophthalmic lens having the composition shown in Table 1 and hydrated and swollen in the same manner as in Example 1 was obtained. This ophthalmic lens was treated with an aqueous calcium chloride solution adjusted to the calcium ion concentration shown in Table 1, and plasmid DNA was supported in the same manner as in Example 1 to perform the above various evaluations.

  The symbols in [Table 1] are as follows.

MOEP methacryloxyethyl phosphate
HEMA 2-hydroxyethyl methacrylate
HPMA Hydroxypropyl methacrylate
NVP N-Vinylpyrrolidone
AAm Acrylamide
EDMA ethylene glycol dimethacrylate

Claims (4)

  A hydrogel ophthalmic lens comprising a gene carried after a treatment in which a hydrogel having a phosphate group is immersed in an aqueous solution containing a divalent metal ion.   The hydrogel ophthalmic lens according to claim 1, wherein the hydrogel having a phosphate group is a gel composed of at least a monomer having a phosphate group and a nonionic hydrophilic monomer.   The hydrogel ophthalmic lens according to claim 1, wherein the divalent metal ion is a calcium ion.   The hydrogel ophthalmic lens according to any one of claims 1 to 3, wherein the gene is plasmid DNA.