WO2019125031A1 - Oral gene carrier and use thereof - Google Patents

Abstract

The present invention relates to an orally-administered gene carrier, and more specifically, to an orally-administered gene carrier having cationic protamine connected to an immunoglobulin Fc region by an SMCC linker, the cationic protamine enabling the condensation of an anionic gene. The orally-administered gene carrier, according to the present invention, enables protamine, which is a protein having cationic properties, to bind to an Fc region and be condensed with a gene having anionic properties, and thus may effectively induce the in vivo expression of the gene, and when orally administered, may enable the gene to be transferred to the small intestine by protecting the gene from a degradation reaction resulting from an immune action of white blood cells and stomach acid, and may enable the half-life of the gene to be relatively long when the gene is expressed in the small intestine, and thus a potential for a long-term treatment effect has been confirmed. Thus, the gene carrier, according to the present invention, is expected to be usefully employed as an orally-administered carrier for various genes.

Description

Oral Gene Delivery and Uses Thereof

The present invention relates to a gene delivery vehicle for oral administration, and more particularly, to a gene delivery vehicle for oral administration in which a cationic protamine capable of aggregating an anionic gene in an immunoglobulin Fc region is linked by an SMCC linker.

The Fc region of the antibody serves to mobilize the immune leukocyte or serum complement molecule to remove damaged cells such as cancer cells or infected cells. The site on the Fc between the Cγ2 and Cγ3 domains mediates the interaction with the neonatal receptor FcRn and its binding recirculates the intracellularly transferred antibody from the endosome into the bloodstream. This process has advantageous antibody serum half-life in the range of 1 to 3 weeks associated with inhibition of kidney filtration due to the enormous size of the full-length molecule. The binding of Fc to FcRn also plays an important role in antibody delivery. Thus, the Fc region plays an essential role in maintaining circulating prolonged serum persistence by circulating antibodies through intracellular trafficking and recycling mechanisms.

On the other hand, although the concept of oral gene therapy has already been proved, nonviral gene delivery through the intestinal tract suffers from many problems because of low expression. There is also a difficulty in effectively controlling intestinal enzymes, microorganisms, and digestive enzymes. However, the Fc receptor (FcRn) transport system has the ability to pass through intestinal epithelial cells, thus solving the problem of absorption efficiency as described above. In particular, the circulation using the Fc receptor has the ability to circulate for the longest time compared to other circulation methods and has a half-life period of 7 to 21 days in human condition, so it can have an effect superior to other types of IgG .

Therefore, in many ongoing clinical trials, in order to increase the half-life of the antibody, a mutation is introduced into the Fc region or a next-generation anti-cancer antibody therapeutic agent is introduced through the Fc domain introduced with mutations to maximize the ADCC (antibody dependent cellular cytotoxicity) (Korean Patent Publication No. 10-2017-0106258). However, the results are still insufficient.

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems. The present inventors have conducted intensive studies on a carrier for efficiently delivering a gene into a body cell. As a result, the present inventors have found that, in order to effectively condense an anionic gene, The present inventors have confirmed that the gene carrier having SMCC linked immunoglobulin Fc region with the protamine having the amino acid sequence shown in SEQ ID NO: 1 has stability against pH and the body enzyme, and confirmed the possibility of use as a gene carrier for oral administration. .

Accordingly, the present invention relates to a DNA encoding a protamine;

Immunoglobulin Fc region; And

And a linker connecting the protamine and the immunoglobulin Fc region.

It is another object of the present invention to provide a method for producing the gene carrier.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating diabetes comprising the gene carrier and the GLP-1 gene combined with the carrier as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

To achieve these and other advantages and in accordance with the purpose of the present invention,

Protamine, which binds to the gene of interest;

Immunoglobulin Fc region; And

And a linker connecting the protamine and the immunoglobulin Fc region.

In one embodiment of the present invention, the immunoglobulin Fc region may consist of the amino acid sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the immunoglobulin Fc region may comprise the nucleotide sequence of SEQ ID NO: 2.

In another embodiment of the present invention, the immunoglobulin Fc region may be derived from any one selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

In another embodiment of the present invention, the immunoglobulin Fc region can be derived from IgG.

In another embodiment of the present invention, the linker may be SMCC (succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate).

In another embodiment of the present invention, the target gene and the gene carrier may be prepared by mixing at a ratio of 1: 5 - 1: 50 wt% (w / w).

In addition, the present invention provides a method for producing the gene carrier, which comprises the following steps.

(a) preparing a protamine-SMCC solution by adding a protamine solution to a solution of succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate;

(b) adding a Cys-Fc solution to the protamine-SMCC solution and culturing to prepare a protamine-SMCC-Fc solution; And

(c) lyophilizing the prepared protamine-SMCC-Fc solution to obtain a gene carrier.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes, which comprises the gene carrier and a GLP-1 (Glucagon Like Peptide-1) gene combined with the carrier as an active ingredient.

In one embodiment of the invention, the diabetes may be type 2 diabetes.

In another embodiment of the present invention, the GLP-1 gene may comprise the nucleotide sequence of SEQ ID NO: 3.

The present invention also provides a method of preventing or treating diabetes, comprising the step of administering the pharmaceutical composition to a subject.

In addition, the present invention provides the use of the pharmaceutical composition for preventing or treating diabetes.

The gene carrier for oral administration of the present invention can effectively induce gene expression in the body by binding protamine, which is a cationic property protein, to the Fc region and condensing it with a gene having an anionic property, and when administered orally, It is possible to transfer the gene to the small intestine by protecting the gene from the degradation due to the immunological action and the half life period is relatively long when the gene is expressed in the small intestine so that the possibility of the long term therapeutic effect is confirmed, Are expected to be usefully useful as carriers for oral administration of various genes.

FIG. 1A shows NMR analysis results to confirm the physical properties of protamine-SMCC-Fc according to the present invention.

FIG. 1B shows FT-IR analysis results of protamine-SMCC-Fc.

FIG. 1C shows the result of confirming whether or not the junction between protamine-SMCC and the Fc region is confirmed using SDS-PAGE.

FIG. 2A shows results of dynamic light scattering (DLS) analysis for confirming the particle size of protamine-SMCC-Fc.

Figure 2b shows SEM imaging results for protamine-SMCC-Fc.

FIG. 2C shows the results of DLS analysis and zeta potential measurements of each complex prepared by mixing DNA and protamine-SMCC-Fc at various weight% (w / w) ratios.

Figure 3 shows the acid stability results of GLP-1 and protamine-SMCC-Fc complexes (DNA + Protamine-SMCC-Fc) under various pH conditions (pH 7.4, 5.6, 1.2).

4A is a SEM image of DNA / protamine-SMCC-Fc (DNA + Protamine-SMCC-Fc) complex.

4B shows AFM analysis results for DNA / protamine-SMCC-Fc (DNA + Protamine-SMCC-Fc) complex.

FIG. 5 shows the result of agarose gel electrophoresis on a complex prepared by varying the ratio of GLP-1 DNA and protamine-SMCC-Fc in order to confirm the junction of DNA / protamine-SMCC-Fc complex.

FIG. 6 shows the results of serum stability and DNase analysis to confirm the gene-protecting ability of the protamine-SMCC-Fc gene carrier according to the present invention by serum and enzymatic degradation.

FIG. 7 shows FcRn-mediated cellular uptake of protamine-SMCC-Fc of the protamine-SMCC-Fc gene transporter according to the present invention in FcRn-positive cells (HT29) and negative cells (KB), respectively.

FIG. 8 shows the cell viability of HT-29 cells treated with the protamine-SMCC-Fc gene transporter at different concentrations.

FIG. 9 shows the results of confirming the cell permeability of the protamine-SMCC-Fc gene transporter.

FIG. 10 shows the results of confirming GLP-1 condensation on the GLP-1 / protamine-SMCC-Fc complex.

FIG. 11 shows the results of in vivo experiments in which the GLP-1-protamine-SMCC-Fc complex was orally administered to the animal model at 4-day intervals and the number of non-fasting blood glucose levels was measured.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have conducted extensive studies on a carrier for efficiently transferring a gene into a body cell. As a result, it has been found that a protein having cationic properties, that is, a protein having an anionic property, And the stability against the enzyme in the body, confirming the possibility of use as a gene delivery vehicle for oral administration, thus completing the present invention.

Accordingly, the present invention relates to a DNA encoding a protamine;

Immunoglobulin Fc region; And

And a linker connecting the protamine and the immunoglobulin Fc region.

As used herein, the term " protamine " is a natural cationic protein rich in arginine, which is found in animal testis, especially in the sperm nucleus of fishes, including salmon, and through association with DNA or dissociation such as histone It is known as a protein involved in genetic information expression. Generally, the molecular weight of protamine extracted from the sperm nuclei of fish is about 4,000 to 10,000, and 70% or more of the constituent amino acids are present as arginine, but the protamine used in the present invention is not limited thereto.

As used herein, the term " immunoglobulin Fc region " refers to the heavy and light chain variable region, the heavy chain constant region 2 (CH2) and the heavy chain constant region (CH1) except for the heavy chain constant region 1 (CH1) 3 (CH3) moiety, and may include a hinge portion in the heavy chain constant region. Also, as long as the immunoglobulin Fc region of the present invention has a substantially equivalent or improved effect as compared with the wild type, the immunoglobulin Fc region may contain a heavy chain constant region 1 (CH1) and / or a heavy chain constant region 1 < / RTI > (CL1). It may also be a region in which some long amino acid sequences corresponding to CH2 and / or CH3 have been removed.

In addition, the immunoglobulin Fc region of the present invention includes a naturally occurring amino acid sequence as well as its sequence derivative (mutant). An amino acid sequence derivative means that at least one amino acid residue in the native amino acid sequence has a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

In the present invention, the immunoglobulin Fc region may consist of the amino acid sequence of SEQ ID NO: 1, and the gene encoding the immunoglobulin Fc region may comprise the nucleotide sequence of SEQ ID NO: 2. At this time, the amino acid sequence shown in SEQ ID NO: 1 or the nucleotide sequence shown in SEQ ID NO: 2 is 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% And most preferably at least 98% sequence homology.

These Fc regions may also be obtained from native forms isolated in vivo in animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs and may be obtained from recombinant animal cells or microorganisms Or derivatives thereof. Here, the method of obtaining from the native form may be a method of separating the whole immunoglobulin from the living body of human or animal, and then obtaining the protein by treating the proteolytic enzyme. When papain is treated, it is cleaved into Fab and Fc, and when pepsin is treated, it is cleaved into pF'c and F (ab) 2. Fc or pF'c can be isolated using size-exclusion chromatography or the like. In the present invention, the immunoglobulin Fc region is preferably a recombinant immunoglobulin Fc region obtained from a microorganism from a human-derived Fc region.

In addition, the immunoglobulin Fc region may be a natural type sugar chain, an increased sugar chain as compared to the native type, and a reduced sugar chain or sugar chain as compared with the native type. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or decrease immunoglobulin Fc sugar chains. At this time, the immunoglobulin Fc region in which the sugar chain is removed from Fc is significantly reduced in binding force with the complement (c1q), and the antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, resulting in unnecessary immune response in vivo Do not. In this regard, forms that are more consistent with the original purpose of the drug as a carrier will be referred to as immunoglobulin Fc regions in which the sugar chain is removed or unglycosylated.

In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof, and most preferably the most abundant ligand binding Derived IgG known to enhance the half-life of the protein.

In the present invention, to effectively condense an anionic gene, protamine having a cationic property is bound to an immunoglobulin Fc region, wherein the linker for binding the protamine and the Fc region is sulfo-SMCC (sulfosuccinimidyl 4 - (N-maleimido-methyl) cyclohexane-1-carboxylate), but not limited thereto, any linker having a property of selectively reacting with both an amine group and a thiol group can be used without limitation.

Accordingly, as another aspect of the present invention, the present invention provides a method for producing the gene carrier comprising the following steps.

(a) preparing a protamine-SMCC solution by adding a protamine solution to a solution of succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate;

(b) adding a Cys-Fc solution to the protamine-SMCC solution and culturing to prepare a protamine-SMCC-Fc solution; And

(c) lyophilizing the prepared protamine-SMCC-Fc solution to obtain a gene carrier.

In the examples of the present invention, in vitro and in vivo analyzes were carried out to confirm the characteristics of the gene carriers prepared by the above method and to verify their availability.

In one embodiment of the present invention, a gene carrier of protamine-SMCC-Fc, in which an immunoglobulin Fc region and protamine are linked, was synthesized using the linker SMCC (see Example 1).

In another embodiment of the present invention, NMR and FT-IR experiments were carried out to confirm the physical properties of the protamine-SMCC-Fc gene transporter. TNBSA analysis was performed to confirm the optimal binding ratio and protamine-SMCC-Fc SDS-PAGE electrophoresis to confirm the junction (see Example 2).

In another embodiment of the present invention, the state of the junction between protamine-SMCC-Fc and DNA is specifically confirmed, and the DNA and the protamine-SMCC-Fc are bonded in a weight ratio of 1:10 or more to form a complex And that the complex was stable at various pH conditions (see Example 3). In addition, in order to confirm efficient gene delivery, the stability and cytotoxicity of the serum were confirmed, and celluar uptake was confirmed to confirm a remarkably high absorption effect in FcRn-positive HT-29 cells.

In addition, the cell permeability of the gene transporter was examined by conducting cell membrane permeability test of the HT-29 monolayer to confirm the cell permeability, and finally, the gene condensation effect was specifically confirmed (see Example 4).

In another embodiment of the present invention, the GLP-1-protamine-SMCC-Fc complex was orally administered to the animal model at 4-day intervals in vivo , and the fasting glucose level was measured. As a result, (See Example 5).

The present inventors confirmed the pH and the enzyme stability of the oral gene carrier according to the present invention through the above results. Since the carrier is absorbed by various organs and has a high absorption rate into the intestinal tract, protamine-SMCC- It is expected that it will have an efficient ability to be used as a gene carrier based on its excellent binding ability. Suggesting that it may be applicable as a carrier of various genes in the future.

Accordingly, as another embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diabetes comprising, as an active ingredient, the gene carrier and GLP-1 (Glucagon Like Peptide-1) gene combined with the carrier do.

In the present invention, the diabetes may be, but is not limited to, type 2 diabetes.

The GLP-1 may comprise the nucleotide sequence of SEQ ID NO: 3, wherein the nucleotide sequence of the GLP-1 is at least 70%, preferably at least 80%, more preferably at least 90% , Preferably 95% or more, and most preferably 98% or more.

As used herein, the term " prevention " means any action that inhibits diabetes or delays the onset of diabetes by administration of the pharmaceutical composition according to the present invention.

As used herein, the term " treatment " refers to any action that improves or alters the symptoms of diabetes by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition according to the present invention contains the gene carrier and the GLP-1 gene as an active ingredient, and may further comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the formulation and include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the intended method, but preferably can be administered orally, Depends on the condition and the weight of the patient, the degree of the disease, the type of the drug, the administration route and time, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level will depend on the type of disease, severity, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and the excretion rate, the type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect of the present invention, the present invention provides a method for preventing or treating diabetes comprising administering the above pharmaceutical composition to a subject.

The term " individual " as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

The present invention also provides the use of the pharmaceutical composition for the prevention or treatment of diabetes.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Preparation of a gene carrier

1-1. Synthesis of protamine-SMCC

dH2O dissolved protamine (5.1kD) (1mole) to (5 mg / mL) gave after stirring for 30 minutes, the pH was adjusted to 7.5 to activate the NH ₂ group. Meanwhile, Sulfo-SMCC (2 moles) was dissolved in a solution (5 mg / mL) containing 300 uL of DMSO and 700 uL of dH ₂ O, followed by addition of Sulfo-SMCC solution dropwise to the protamine solution prepared above , At which time it was stirred continuously at room temperature for 24 hours. The reaction mixture was then dialyzed against distilled water (MWCO: 3.5-5 kD) for 2 days and lyophilized to obtain protamine-SMCC.

1-2. Production of protamine-SMCC-Fc

First, protamine-SMCC was dissolved in PBS at pH 6.5-7.4, and the solution was adjusted to a concentration of 3 mg / mL while being stirred for 30 minutes. Then, Cys-Fc was dissolved in PBS (1 mole) And the solution was added dropwise to the protamine-SMCC solution while stirring at room temperature for 1 hour. Thereafter, the cells were cultured overnight at 4 ° C (12 hours) without further agitation, and then dialyzed against dH ₂ 0 for 24 hours and lyophilized to obtain protamine-SMCC-Fc.

Example 2. Identification of the physical properties of protamine-SMCC-Fc

2-1. NMR and FT-IR analysis

NMR and FT-IR experiments were conducted to confirm the physical properties of the protamine-SMCC-Fc obtained from Example 1, and the results are shown in FIGS. 1A and 1B. More specifically, FIG. 1A shows NMR spectral results of protamine (No. 1), SMCC (No. 2), Protamine-SMCC (No. 3) and Protamine-SMCC-Fc (No. 4) (a), protamine-SMCC (b) and protamine-SMCC-Fc (c).

2-2. TNBSA analysis

TNBSA (2,4,6-Trinitrobenzene Sulfonic Acid) analysis was performed to determine the optimal conjugation ratio of protamine and SMCC.

As a result, it was confirmed that the ratio of 0.4 nm SMCC to 1 nm protamine was optimal, as shown in Table 1 below. Specifically, when the molar ratio of protamine-SMCC was 1: 2 and 1: 3, There was no difference, and the molar ratio was optimized to 1: 2.

Feed mole ratio (Protamine: SMCC)	Conjugation ratio
1: 2	1: 0.4
1: 3	1: 0.4

2-3. SDS-PAGE electrophoresis

In addition, as shown in Fig. 1C, when protamine-SMCC-Fc was conjugated through SDS-PAGE using 12% acrylamide gel, the protamine-SMCC- It was confirmed that the molecular weight (75 kDa) of the Fc sample was larger.

2-4. Particle size analysis

Dynamic Light Scattering (DLS) analysis and scanning electron microscope (SEM) image analysis were performed to confirm the particle size of Protamine-SMCC-Fc according to the present invention.

As a result, as shown in the DLS analysis results of FIG. 2A, the protamine-SMCC particle size was 105.3 nm, while the size of protamine-SMCC-Fc was 161.1 nm. As a result of SEM imaging analysis, it was confirmed that the protamine-SMCC-Fc particles showed a monodispersed shape as shown in FIG. 2b, and the particle size was measured similarly to the DLS analysis result. The measured protamine-SMCC-Fc particle sizes as a result of DLS and SEM analysis are summarized in Table 2 below.

name	DLS size	SEM size
Protamine-SMCC	105.3 nm	90 nm
Protamine-SMCC-Fc	161.1 nm	120 nm

Example 3. Electrostatic interaction study.

3-1. Measure particle size and zeta potential

The particle size was measured by DLS analysis on the complex of GLP1 DNA and protamine-SMCC-Fc transporter mixed at various ratios (1: 1, 1: 5, 1:10, 1:15, 1:25) (Zeta potential) analysis.

As a result, as shown in FIG. 2C, when the ratio of the GLP1 DNA to the protamine-SMCC-Fc transporter is more than 1:15, the particle size does not decrease even if the zeta potential is similar, It was confirmed to be dense to form a complex.

3-2. pH stability analysis

Next, we tried to verify the stability of GLP1 and protamine-SMCC-Fc complexes in various pH environments because the pH of each organ is different when orally administered. For this purpose, each complex of GLP1 DNA and protamine-SMCC-Fc transporter was mixed at various ratios under the conditions of pH 7.4, 5.6 and 1.2, and analyzed by DLS for time (0, 3, 6, Were measured.

As a result, as shown in FIG. 3, the complex was found to be stable at pH 7.4, 5.5, and 1.2, and the particle size was reduced to 1:15 ratio and the 1:25 ratio complex was similar in size The optimization ratio of GLP1 DNA and protamine-SMCC-Fc transporter was confirmed to be 1:15 once again.

3-3. SEM and AFM analysis

First, in order to confirm the form of the complex in which the GLP-1 DNA and the protamine-SMCC-Fc transporter were mixed, the complex was prepared at a ratio of 1:10 and 1:15, respectively, and SEM image analysis was performed. 4A.

In addition, as shown in FIG. 4B, when the DNA: protamine-SMCC-Fc was produced at a ratio of 1:15 as compared with naked DNA, the atomic force microscope (AFM) And it was again confirmed that the above ratio is the optimum bonding ratio.

3-4. Agarose gel electrophoresis

The complexes in which the GLP-1 DNA and the protamine-SMCC-Fc transporter were mixed at various ratios were subjected to agarose gel electrophoresis to confirm the formation of the complex. For this, GLP-1 DNA alone, complexes prepared with various ratios (1: 1, 1: 2, 1: 5, 1:10, 1:12, 1:15, 1:25, 1:30) + 1% agarose gel electrophoresis was performed using a GLP-1 DNA sample.

As a result, as shown in FIG. 5, it was confirmed that the band disappears from the ratio of GLP-1: protamine-SMCC-Fc of 1:10, whereby GLP-1 binds compactly with protamine-SMCC-Fc, .

Example 4. in vitro  Research

4-1. Serum Stability Test and DNase Analysis

Serum stability and DNase analysis were carried out to verify the ability of the oral type gene transporter according to the present invention to protect the gene and the enzyme against degradation.

As a result, as shown in FIG. 6, the degradation started to proceed in serum after 8 hours in the case of GLP-1 gene alone, whereas in the case of GLP-1 + protamine-SMCC-Fc, Was found to protect the gene from serum degradation.

4-2. FcRn mediated cellular uptake study

Cellular uptake studies of protamine-SMCC-Fc in HT-29 (FcRn +) and KB (FcRn-) cells were conducted through confocal microscopy to confirm the interaction between FcRn and Fc and absorption in FcRn- Respectively.

As a result, as shown in Fig. 7, it was confirmed that the pratamine-SMCC-Fc showed significantly higher cellular uptake in FcRn-positive HT-29 cells compared with KB cells.

4-3. Cytotoxicity check

Next, to examine cytotoxicity by protamine-SMCC-Fc, HT-29 cells were treated with protamine-SMCC-Fc at various concentrations (5, 10, 25, 50, 100 ug / ml) After 72 hours, cell viability was measured by MTT assay.

As a result, as shown in Fig. 8, it was confirmed that the cell viability was maintained to be high at all the concentrations up to 72 hours as compared with the control (control), and the gene carrier according to the present invention did not induce cytotoxicity in the cells .

4-4. Confirmed epithelial transport of Fc

In order to confirm the cell permeability of the gene carrier according to the present invention, 3 x 10 ⁵ cells / well / 5.5 mL of HT-29 cells were cultured for 7 days and recovered. The medium was washed with HBSS + 0.05% BSA + 10 mM MES pH 6.0 Chamber) and 10 mM HEPES, pH 7.4 (basolateral chamber), respectively. The basolateral portion was recovered after treatment with different samples at different time intervals for fluorescence analysis.

As a result, as shown in Fig. 9, it was confirmed that the cellular uptake increased 5-fold in the gene delivery body compared to DNA alone after 24 hours.

4-5. GLP-1 Condensation Analysis

The EtBr displacement assay was performed to confirm GLP-1 condensation on the GLP-1 / protamine-SMCC-Fc complex according to the present invention.

As a result, GLP-1 alone showed higher fluorescence as compared to GLP-1 / protamine-SMCC-Fc complexes in different ratios as shown in FIG. 10, indicating that GLP- Lt; RTI ID = 0.0 > complete condensation. ≪ / RTI > From the above results, it has been confirmed that the gene carrier according to the present invention is absorbed by various organs, and since the absorption rate of the intestinal tract is high, protamine-SMCC-Fc has excellent binding force with various organs, It is expected to show ability. It was confirmed that it could be applied to various gene carriers in the future.

Example 5. in vivo Research

In addition to the results of Example 4 above, in vivo experiments were conducted to verify the therapeutic effect of GLP-1 / protamine-SMCC-Fc complex according to the present invention on diabetes. For this, we used the BKS.Cg + / + Lepr db / db mouse model, a type 2 diabetes model, in which the insulin level started to increase from 10 to 14 days after birth at 4 to 5 weeks And it is a model having the feature that the obesity starts and hyperlipemia occurs and the rate of glucosuria is 100% after 10 weeks. For the experiment, the complex was orally administered to the mouse model at 4-day intervals, and the control group was administered with PBS and daily non-fasting blood glucose levels were measured for up to 32 days.

As a result, as shown in FIG. 11, when the GLP-1-protamine-SMCC-Fc was administered, unlike the control group, the non-fasting blood glucose level dropped to the normal range. To the normal level.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

The gene carrier for oral administration according to the present invention can be used as an effective oral gene delivery vehicle capable of stably transferring gene to the small intestine by protecting the gene from the body environment. The gene carrier having the GLP-1 gene loaded on the gene carrier And thus the complex can be usefully used in the field of the prevention or treatment of diabetes.

Claims (13)

1. Protamine, which binds to the gene of interest;

   Immunoglobulin Fc region; And

   And a linker connecting the protamine and the immunoglobulin Fc region.
2. The method according to claim 1,

   Wherein the immunoglobulin Fc region comprises the amino acid sequence of SEQ ID NO: 1.
3. The method according to claim 1,

   Wherein the immunoglobulin Fc region comprises the nucleotide sequence of SEQ ID NO: 2.
4. The method according to claim 1,

   Wherein the immunoglobulin Fc region is derived from any one selected from the group consisting of IgG, IgA, IgD, IgE and IgM.
5. 5. The method of claim 4,

   Wherein said immunoglobulin Fc region is derived from IgG.
6. The method according to claim 1,

   Wherein the linker is SMCC (succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate).
7. The method according to claim 1,

   Wherein the target gene and the protamine-SMCC-Fc gene carrier are mixed at a ratio of 1: 5 - 1: 50 wt% (w / w).
8. A method for producing a gene carrier according to claim 1, comprising the steps of:

   (a) preparing a protamine-SMCC solution by adding a protamine solution to a solution of succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate;

   (b) adding a Cys-Fc solution to the protamine-SMCC solution and culturing to prepare a protamine-SMCC-Fc solution; And

   (c) lyophilizing the prepared protamine-SMCC-Fc solution to obtain a gene carrier.
9. A pharmaceutical composition for the prevention or treatment of diabetes comprising the gene carrier of any one of claims 1 to 7 and a GLP-1 (Glucagon Like Peptide-1) gene combined with the carrier as an active ingredient.
10. 10. The method of claim 9,

    Wherein the diabetes is type 2 diabetes.
11. 10. The method of claim 9,

    Wherein the GLP-1 gene comprises the nucleotide sequence of SEQ ID NO: 3.
12. A method of treating diabetes mellitus comprising administering to a subject a pharmaceutical composition comprising the gene carrier of any one of claims 1 to 7 and a GLP-1 (Glucagon Like Peptide-1) Prevention or treatment.
13. A pharmaceutical composition comprising the gene carrier of any one of claims 1 to 7 and the GLP-1 (Glucagon Like Peptide-1) gene combined with the carrier as an active ingredient for the prophylaxis or treatment of diabetes.

Images