KR20030068337A - Eukaryotic expression vector for human gene therapy - Google Patents

Abstract

PURPOSE: Provided is a pJDK vector which involves high cell transfection efficiency and high recombinant protein expression efficiency, and is available in making an adeno-associated virus vector for gene therapy. CONSTITUTION: A pJDK vector(KCCM 10343) contains a cytomegalovirus (CMV) promoter, an inverted terminal repeat (ITR) of an adeno-associated virus, a bovine growth hormone (BGH) polyadenylation signal, and kanamycin resistance gene. A target gene is expressed by introducing the pJDK vector in a cell. The cell is an animal cell or derived from animal. A recombinant adeno-associated virus is produced using the pJDK.

Description

Eukaryotic expression vectors for gene therapy {EUKARYOTIC EXPRESSION VECTOR FOR HUMAN GENE THERAPY}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to eukaryotic expression vectors for gene therapy, and more particularly, to pJDK vectors which are excellent in intracellular gene transfection efficiency and expression efficiency and can be used for viral vector production.

[Private Technology]

Gene therapy, which began in earnest for the treatment of congenital genetic diseases since 1990, has been extended to the treatment of all diseases.

The main reason why gene therapy technology is not popularized / industrial is that the vectors that are being used cannot deliver genes safely and efficiently. In particular, it does not exhibit a recombinant protein efficiency that can exhibit a disease treatment effect, gene expression is short-term, and expression stability has not been established.

The key to gene therapy lies in the therapeutic genes that actually produce a therapeutic effect, the vectors that deliver genes to the human body (vectors), and in vivo gene delivery technologies.

Vectors used in actual clinical trials can be classified into two types: biological vectors (viruses) and physicochemical vectors (liposomes, naked DNA, etc.).

Of the 2000 worldwide genetic therapy trial data, 70% of the trials reported using viruses, of which 54% were retroviruses. However, viral vectors have problems such as the risk of infection by wild-type viruses, genetic mutations due to intermittent insertion on host chromosomes, and the development of immune responses in the body to viral proteins.

Naked DNA refers to the vector itself, which is performed by injecting exposed DNA into the body in gene therapy. Gene therapy using exposed DNA has the advantage of being able to easily recombine the therapeutic gene into an exposed vector, secure large amounts of vector DNA, require less preparation and procedure time, and have little toxicity on its own. Rizzuto G et al., Proc Natl Acad Sci USA 1999; 96: 6417-6422)

However, currently used exposure vectors have very low gene transfer efficiency in vivo (within 1%), so it is difficult to expect therapeutic effects of therapeutic genes, and expression efficiency of recombinant proteins is also very low. Generally used exposure vector contains the ampicillin resistance gene, and the ampicillin in the medium is added during the selection and mass growth of the exposure vector. When such ampicillin is introduced in vivo with an exposure vector, it can act as a toxic factor in patients allergic to penicillin. Also, general exposure vectors could not be used for the preparation of adeno-associated viruses.

Therefore, an object of the present invention is to provide a vector with high gene transfer efficiency in vivo.

It is also an object of the present invention to provide a safe vector in vivo.

It is also an object of the present invention to provide a vector usable for the preparation of adeno-associated viruses that can be used as a viral delivery vector.

In addition, an object of the present invention is to provide a vector with high expression efficiency of the therapeutic gene.

Figure 1 shows the manufacturing process of the pJDK vector,

Figure 2 shows a genetic map of the pJDK vector,

3 is a graph quantifying the expression level of CAT in HeLa cells (a) and C2C12 cells (b) injected with pcDNA3.1-CAT or pJDK-CAT,

Figure 4 is a measure of the CAT expression after the muscle injection of pcDNA3.1-CAT or pJDK-CAT, a is a graph analyzing the expression of CAT when the electric shock was not applied after the muscle injection, b is a post-injection muscle This graph shows the analysis of CAT expression levels after impact.

In order to achieve the above object, the present invention provides a CMV promoter, an inverted terminal repeat (ITR) of adeno-associated virus (hereinafter referred to as 'AAV'), a Bovine Growth Hormome (BGH) polyadenylation signal, and kanamycin. A pJDK vector (KCCM 10343) comprising a resistance gene is provided.

The present invention also provides a method of expressing a gene of interest by introducing a pJDK vector containing the gene of interest in a cell.

The present invention also provides adeno-associated viruses produced using the pJDK vector.

Hereinafter, the present invention will be described in detail.

Eukaryotic expression vector pJDK of the present invention comprises the following configuration, more specific gene map is shown in FIG.

(1) CMV (cytomegalovirus) promoter

(2) Inverted terminal repeat (IRT) of adeno-associated virus (AAV)

(3) kanamycin resistance gene

(4) kanamycin resistance gene

The efficiency of recombinant protein expression of the vector depends on the promoter. Promoters include viral promoters, promoters of housekeeping genes that always induce gene expression in eukaryotic cells, tissue-specific promoters that determine gene expression in specific tissues. The CMV virus is the strongest promoter of the viral promoters.

The ITR of the AAV has a palindrome structure of 145 bp and is involved in replication of the AAV and insertion of a host chromosome. Although ITR has been reported to increase gene expression of eukaryotic expression vectors, no studies have been conducted on whether the gene expression is increased and its mechanism of action when used in exposed DNA.

The kanamycin resistance gene is used as a selection factor for mass production of pJDK vectors and for the production or selection of transformants and recombinant vectors using the pJDK vector. The kanamycin is safe in vivo, and even if kanamycin is contaminated in the pJDK vector, it does not act toxic to the living organism. The safety of kanamycin is specified in the Gene Therapy Guidelines of the US Food and Drug Administration and the Korea Food and Drug Administration.

The pJDK vector of the present invention is composed of a total of 3,354 bp, the nucleotide sequence thereof is described as SEQ ID NO: 1, pJDK gene map is shown in FIG. The origin and origin of each part of the pJDK is shown in Table 1 below.

Vector name of origin CMV promoter pUHD15-1 Multiple Cloning Sites pBSIISK Poly-A Signal Rc / CMV Kanamycin resistance gene pVAX1 ori pVAX1 ITR pSUB201 Plasmid Basic Structure pVAX1

The base sequence of pJDK is shown in SEQ ID NO: 1, and the detailed restriction map is shown in FIG.

The pJDK vector can be used as a carrier for transferring and expressing an external gene in a cell, and the pJDK vector is transfected into 293 cell lines simultaneously with a Rep-Cap expression vector and an adenovirus gene expression vector to generate ITR. Recombinant adeno-associated viruses can be prepared. Lab-cap expression vectors provide genes necessary for the proliferation of adeno-associated viruses and for the preparation of surface proteins. The 293 cell line is a host cell that expresses the genes so that adeno-associated viruses are packaged and produced.

The pJDK vector of the present invention not only has high intracellular transfection efficiency and recombinant protein expression efficiency (FIG. 3), but also shows excellent transfection efficiency when electroporated after transfection (FIG. 4). Preferred application of electricity is to carry out a current of 100 to 140 v / cm, and most preferably to carry out a current of 125 V / cm 8 times at a rate of once per second for 50 msec.

The pJDK vector of the present invention can be used by inserting an external gene to transfer and express an external gene, to produce a recombinant adeno-associated virus, gene therapy and the like.

The present invention also provides a method of expressing a gene of interest by introducing a pJDK vector containing the gene of interest in a cell. The gene of interest can be any conventional protein, for example, enzymes, cytokines and the like. The cell is preferably an animal cell or an animal derived cell.

In addition, the method for expressing the gene of interest of the present invention further comprises introducing the pJDK vector into the cell and applying electricity to the cell. Electrical application is repeated 8 times with a current of 125 V / cm at a rate of once per second for 50 msec.

The present invention also provides a recombinant adeno-virus produced using the pJDK vector. The pJDK vector can be transfected simultaneously to a 293 cell line with a Rep-Cap expression vector and an adenovirus gene expression vector to produce a recombinant adeno-associated virus using ITR. The lab-cap expression vector is a vector that provides genes necessary for the propagation of adeno-associated viruses and the preparation of surface proteins, and the 293 cell line expresses the genes so that the adeno-associated viruses are packaged and produced. The recombinant adeno-virus produced was proliferating in 293 cells.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1 Preparation of ACP Vectors

(1) Preparation of AAV-CMV Vector

1. Preparation of CMV Promoter and Multiple Cloning Site (MCS)

A 766 bp HCMV promoter fragment was obtained by cleaving around the HCMV promoter of the pUHD15-1 vector (FIG. 1A- (1), kind gift from H. Bujard) with an XhoI / EcoRI enzyme. HCMV fragments were inserted into a pBSII SK (FIG. 1A- (2), Stratagene) vector treated with XhoI / EcoRI, digested with XhoI / XbaI enzyme to obtain HCMV + MCS cassette, and then treated with Klenow enzyme to blunt. end) into sections.

2. preparation of a basic plasmid comprising ITR;

pSUB201 (FIG. 1A- (3), kind gift from Dr. Samulski) was cut with XbaI, and sections of about 4KB containing ITR were separated. Sections were blunt end treated with Klenow enzyme.

3. Preparation of AAV-CMP Vectors

The fragments containing the HCMV + MCS cassette and ITR prepared above were linked to prepare an AAV-CMP vector (FIGS. 1A-4).

(2) Preparation of ACP Vector

1. Transcription Factor Cloning and ACP Preparation

The Rc / CMV (FIG. 1b- (5), Invitrogen) vector was cut with BamHI enzyme, and then a fragment containing a portion of polylinker and a bovine growth hormone (BGH) polyA was inserted into the BAMHI-treated AAV-CMV vector. 1b- (6). Subsequently, the fragments containing the restriction enzyme cleavage site were removed by cutting with EcoRI, and then linked to prepare an ACP vector (FIGS. 1B- (7)).

Example 2: Preparation of pJDK Vectors

The ACP vector of Example 1 showed high gene expression in vitro and in vivo , but had an ampicillin gene as a selection factor. Therefore, ampicillin contained in the medium for selection of transformants may cause an allergic reaction such as shock in a patient with penicillin allergy. Thus, a new vector was prepared by replacing the ampicillin resistance gene of ACP with kanamycin resistance gene.

The pVAX1 vector (FIG. 1C- (8), Invitrogen) was cut at the HincII / XcmI site and fragments containing the kanamycin resistance gene were blunt-ended with T4 DNA polymerase. The ACP vector was cut with PvuII and the 1.49 kb fragment of ITR-HCMV-MCS-polyA-ITR was isolated and then linked to the fragment containing the kanamycin resistance gene to prepare pJDK (FIG. 1C- (9)).

pJDK was deposited and given KCCM10343. Vector map of pJDK is shown in Figure 2, the nucleotide sequence is shown by SEQ ID NO: 1.

Example 3 Verification of Transfection Efficiency of pJDK Vectors

PCDNA3.1-CAT and pJDK-CAT were prepared by inserting the CAT gene into the control vectors pcDNA3.1 and pJDK.

(1) gene injection

HeLa cells (ATCC; CCL-2), C2C12 (ATCC; CRL-1772) cells, which are skeletal myoblasts of C3H mice, were placed in a 12-well plate in a 12-well plate under DMEM / 10% FBS (fetal bovine serum, manufactured by Gibco BRL). Transfection experiments were performed one day after spreading to ^four .

2 μg each of pcDNA3.1-CAT and pJDK-CAT and FuGENE6 (manufactured by Roche) were mixed in a microtube at a ratio of 1: 3 (v / v), and reacted at room temperature for 15 minutes, followed by lipoplex. Dropped into culture of HeLa cells or C2C12 cells. After 48 hours of incubation, the cells were collected and CAT quantified.

The cells were washed with 4 ° C. PBS and then treated with 200 ul of CAT lysis buffer. The cells were scraped with a scraper and allowed to react for 30 minutes on ice. After centrifugation at 13,000 rpm for 20 minutes at 4 ℃ supernatant was taken to quantify the expressed CAT.

(2) CAT expression quantification

The cells were placed in CAT Lysis buffer (manufactured by Boehringer Mannheim) and homogenized at 4 ° C. The suspension was collected by centrifugation at 4 ° C. and 12,000 rpm for 5 minutes. After diluting the suspension by 1/10 to 1/100, the amount of CAT was quantified using a CAT ELISA kit (manufactured by Boehringer Mannheim).

Data analysis was performed with InStat program (version 2.02). The comparison between the two groups was analyzed by the Mann-Whitney test. When the p- value was less than 0.05, statistical significance was recognized, and the data were expressed as mean ± standard error.

Figure 3 is a graph quantifying the expression level of CAT in HeLa cells (a) and C2C12 cells (b) injected with pcDNA3.1-CAT or pJDK-CAT. CAT expression by pJDK in HeLa cells was 28-fold higher than pcDNA3.1 (pcDNA3.1 vs pJDK = 12,830 ± 86 vs 363,507 ± 22,130 pg / ml, p = 0.029), and CAT expression in C2C12 cells was pcDNA3. 12 times higher than 1 (pcDNA3.1 vs pJDK = 2,487 ± 258 vs 29,900 ± 2,176 pg / ml, p = 0.029)

Therefore, the pJDK of the present invention not only has high transfection efficiency, but also the expression efficiency of the recombinant protein is much better than that of the conventional vector. This is thought to be due to the interaction between the CMV promoter, a potent viral promoter, the ITRs known to increase gene expression of eukaryotic expression vectors, and the polyadenylation signals of BGH known to increase the stability of transcriptional RNA.

Example 4: Animal Experiment

(1) gene injection

Animal experiments were conducted according to the Animal Experiment Rules of the US National Institute of Health ("Guide for the care and use of laboratory animals, NIH publication No. 85-2, revised 1996) and Samsung Medical Center, Seoul National University)

A 5 week old female BALB / c-AnNCrj mouse was used. Mice were anesthetized with Ketamine and Xylazine, and 10 ug DNA / 30 ul NaCl was injected into the tibialis anterior muscle using a 30-gauge insulin syringe (manufactured by Beckton Dickinson). The P10 tube was inserted into the needle so that the needle did not enter more than 3 mm. Thirty seconds after the gene was injected, an electric shock was applied using an electroporator (ECM 830, manufactured by BTX Division of Genetronics). The electric shock was impacted eight times with an intensity of pulse parameter 125 v / cm, 50 msec, 1 Hz. Electrode (Electrode, Tweezertrodes, manufactured by BTX Division of Genetronics) is a forceps shape, the electric shock was applied to the skin on both sides of the gene injection section. After 7 days, the muscle was harvested at the expense of mice to quantify CAT expression.

Figure 4 is a measure of the CAT expression after the muscle injection of pcDNA3.1-CAT or pJDK-CAT, a is a graph analyzing the expression of CAT when the electric shock was not applied after the muscle injection, b is a post-injection muscle This graph shows the analysis of CAT expression levels after impact. CAT expression by pJDK was three-fold higher than pcDNA3.1 (pcDNA3.1 vs pJDK = 1,015 ± 342 vs 3,119 ± 1,846 pg / ml, p = 0.691). The expression of CAT by pJDK was 34-fold higher than that of pcDNA3.1 (pcDNA3.1 vs pJDK = 4,650 ± 1,461 vs 156,100 ± 87,680 pg / ml, p = 0.010). -CAT was measured to increase the expression of CAT 5 times by electric shock, and pJDK was measured to increase the expression of CAT 50 times.

Therefore, the pJDK vector of the present invention not only has excellent gene transfer efficiency and expression efficiency in vivo, but also greatly increases expression efficiency by application of electricity.

As mentioned above, the pJDK vector of the present invention is not only high in intracellular transfection efficiency and recombinant protein expression efficiency, but also can be used as an adeno-associated viral vector and thus is useful for gene therapy.

Claims (6)

PJDK vector (KCCM 10343) comprising CMV promoter, inverted terminal repeat (IRT) of adeno-associated virus, Bovine Growth Hormone (BGH) polyadenylation signal, kanamycin resistance gene. A method of expressing a gene of interest by introducing a pJDK vector of claim 1 containing a gene of interest into a cell. The method of claim 2, wherein the cell is an animal cell or an animal derived cell. The method of claim 2, further comprising introducing the pJDK vector into the cell and applying electricity to the cell. Recombinant adeno-associated virus produced using the pJDK vector of claim 1. The method of claim 5, wherein the recombinant adeno-associated virus (a) a CMV promoter; (b) an inverted terminal repeat (ITR) of adeno-associated virus; And (c) Bovine Growth Hormome (BGH) polyadenylation signal Recombinant adeno-associated virus comprising a.