KR101390967B1 - Retroviral vectors with improved safety - Google Patents

Abstract

The present invention relates to a recombinant retroviral vector derived from murine leukemia virus (MLV), which deletes only the optimal U3 region in consideration of safety and viral activity, and more specifically relates to a recombinant retrovirus vector derived from MLV LTR ) Derived MLV-derived recombinant retroviral vector in which the nucleotide numbers x to y of the U3 portion are missing (where x is 7800 to 7900 and y is 8100 to 8260).

The viral vector of the present invention has low possibility of activating a peripheral gene of a target cell and is safe and can be usefully used for gene therapy because the viral titer and the expression rate of a target gene are kept high.

Description

BACKGROUND ART Retroviral vectors having improved safety {RETROVIRAL VECTORS WITH IMPROVED SAFETY}

1 , The structure of the plasmid prepared in Example 1 is shown,

FIG. 2 shows the results of confirming the expression level of CAT mRNA by Northern blotting after plasmid prepared in Example 1 was transfected into K562 cells.

3 shows an LTR removal process in the second embodiment,

Fig. 4 shows deletion sites of the plasmid-type retrovirus vectors pI-D1 to pI-D5 prepared in Example 2,

Fig. 5 shows the structure of pI-LND-n in Example 3,

6A and 6B show the structure of pC-LND-GFP-n of Example 3 and production and transfection of retrovirus in 293T cells using the same, respectively.

7 is a graph showing FACS analysis of gene expression in HT1080 cells of C-LND-GFP-n retrovirus obtained from 293T cells in Example 3. In the figure, MFI means mean fluorescence intensity,

FIGS. 8A and 8B show the structure of pC-LND-GFP-n of the linearized Example 3 and the production and transfection of the retrovirus in the production cell line PG13 using the same.

FIG. 9 shows the results of FACS analysis of gene expression in HT1080 cells of C-LND-GFP-n retrovirus obtained from the production cell line in Example 3,

Figure 10 The results of FACS analysis confirmed the gene expression in K562 cells of C-LND-GFP-n retrovirus obtained from the production cell line in Example 3.

The present invention relates to a recombinant retrovirus vector derived from murine leukemia virus (hereinafter referred to as MLV), which enhances safety while maintaining the activity of the virus and the expression efficiency of the target gene.

Retrovirus vectors have been widely used for the treatment of diseases, particularly genetic diseases, which require continuous gene expression, since they are inserted into the chromosome of the target cell and the cells can be transferred to the offspring cells without disappearance of the gene. Most retroviral vectors used in gene therapy use the MLV genome as a basic genome. Such retroviral vectors require many improvements in terms of safety, gene expression level, and viral titer.

In particular, since the process of inserting the vector into the chromosome of the target cell occurs nonspecifically, it can cause fatal side effects when inserted around the important gene. For example, LTR (long terminal repeat) of retroviral vectors has a transcriptional regulatory sequence that can promote the expression of a peripheral gene. When a retroviral vector is inserted around a tumor gene, expression of the oncogene is increased by the LTR And can cause cancer. As an example, in early 2003, leukemia developed in some patients who received gene therapy with retroviral vectors for the treatment of X-linked severe combined immunodeficiency (X-SCID), a congenital immune deficiency syndrome. Later, it was reported that the retroviral vector used for gene therapy was associated with leukemogenesis (Hacein-Bey-Abina Et al., Science 302: 415-9, 2003) The possibility of insertion mutation in gene therapy using retroviral vectors has emerged as an important issue.

A commonly proposed method for lowering the side effects of such insertion mutations is to remove the transcriptional control sequences present in the LTRs of the retroviral vector. The LTR consists of three parts, U3, R and U5, of which U3 (corresponding to nucleotides 7816-8264 based on NCBI registration number J02255; SEQ ID NO: 25) contains upstream control regions and repeat sites an enhancer including a direct repeat; And a promoter comprising CAAT box and TATA box (Sun et al ., J Virol 69: 4941-9, 1995; and Wahlers et al., Mol Ther 6: 313-20, 2002). Thus, removal of the U3 portion from the retroviral vector can prevent activation of the surrounding gene, and cases of U3 removal in various ways have been reported. For example, Yu et al. (Yu et al., Proc Natl Acad Sci USA 83: 3194, 1986) reported that from the PvuII site to the SacI site of the 3'LTR of the MLV vector (corresponding to nucleic acid 7938 to 8233 according to NCBI registration number J02255) Respectively. In this case, the direct repeat of the enhancer portion and the CAAT box of the promoter portion are removed. In addition, Yee et al. (Yee et al., Proc Natl Acad Sci USA 84: 5197, 1987) removed the enhancer, CAAT box, and TATA box site (corresponding to NCBI registry number J02255, nucleic acid 7909 to 8240) in the LTR of the MLV vector I made a vector.

Although the U3-deleted vector has a reduced ability to activate the peripheral gene, there is still a possibility that the peripheral gene is activated by an internal promoter for expression of the desired gene. Therefore, an insulator sequence for blocking the influence of the internal promoter may be inserted (Ramezani et al., Mol Ther. 14: 245-54, 2006), and the gene expressed by the internal promoter does not stop at the transcription termination position (Ramezani et al., Mol Ther. 14: 245-54, 2006) in order to prevent transcription and expression of peripheral genes.

However, in order to increase the safety of the vector, if the U3 region is removed or the inserter sequence or polyadenylation sequence is added, the virus titer decreases.

Therefore, the present inventors have intensively studied the development of a vector that does not lower the potency of the virus and the expression rate of the target gene while securing the safety of the retrovirus vector. As a result, the inventors have found that, Confirming the optimal deletion site of LTR, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a retrovirus vector that enhances safety while maintaining the activity of a virus and the expression efficiency of a target gene.

In order to accomplish the above object, the present invention provides a method for producing a murine leukemia virus (MLV), wherein the nucleotide number x to y of the U3 portion of LTR (long terminal repeat) of the murine leukemia virus (MLV) 7900 and y is 8100 to 8260) to provide a MLV-derived recombinant retroviral vector.

Further, the present invention relates to (1) the MLV-derived recombinant retrovirus vector; And (2) an expression cassette comprising a selectable marker gene, a heterologous promoter operably linked thereto, and a polyadenylation signal.

Also, the recombinant retrovirus producing cell line comprising the MLV-derived recombinant retroviral vector or the recombinant plasmid; A method of culturing the production cell line to produce recombinant retrovirus; And a cell transfected with the recombinant retrovirus produced from the production cell line.

Finally, the invention relates to a heterologous gene and a heterologous promoter operably linked thereto; And a recombinant retrovirus (wherein x is 7800 to 7900 and y is 8100 to 8260) comprising the genome of an MLV-derived recombinant retrovirus deleted from the nucleotide numbers x to y of the U3 portion of the LTR of MLV As an active ingredient, a pharmaceutical composition for gene therapy.

Hereinafter, the present invention will be described in detail.

The MLV-derived recombinant retrovirus vector of the present invention is characterized in that the viral titer can be maintained while some of U3 of LTR is deleted to improve safety.

Specifically, in the retrovirus vector of the present invention, deletion sites of the U3 are deleted from the nucleotide numbers x to y based on NCBI registration number J02255, wherein x is 7800 to 7900, y is 8100 to 8260, More preferably, x is 7840 to 7860 and y is 8100 to 8244. The most preferred deletion sites are those in which x is about 7847 and y is 8113 to 8208.

The term "retrovirus" in this application refers to an RNA virus that has a reverse transcription process in the life history. The genomic RNA of the retrovirus is converted into double stranded DNA by reverse transcriptase, and the double stranded DNA virus can be inserted into the chromosome of the infected cell. Once inserted into the cell's chromosome, it is called a "pro-virus", which can be used as a template for RNA polymerase and expressed as RNA.

In the present invention, the term "vector" refers to any genetic material capable of transferring a target gene into a cell. Examples of the genetic material include plasmids, phage, viruses, and cosmids.

A "retroviral vector" refers to a genetic material derived from a retrovirus, and a retroviral vector includes a cis sequence required for viral particle production. The cis sequence includes the packaging sequence and primer binding site necessary for the genomic RNA of the virus to be packaged into the viral particle, but the viral structural protein genes, gog, pol, and env . Virus structure proteins for viral particle production are provided from packaging constructs.

The heterologous gene inserted into the retroviral vector of the present invention for the purpose of expression thereof may be a biologically active protein or polypeptide, an antigenic protein or polypeptide, a nucleotide sequence encoding a therapeutic protein or a polypeptide have. In addition, the heterologous gene may be a nucleotide sequence encoding anti-sense RNA, siRNA (small interfering RNA), ribozyme, or the like. Preferably selected from the group consisting of enhanced green fluorescence protein (eGFP), luciferase, gp91-phox, iduronate-2-sulfatase, adenosine deaminase, IL-2 receptor gamma, Antigens, antigens, antigens, immunosuppressive agents, anti-sense RNAs, siRNAs, ribozymes, and even more preferably, cytokines (IL-2, IL-6, IL-12, IL-13 and IL-15) Enhanced green fluorescent protein or gp91-phox can be used.

If the U3 portion is deleted as in the present invention, a heterologous internal promoter must be separately inserted in order to express the heterologous gene of interest. The heterologous inner promoter is for promoting the expression of the target gene when the retroviral vector of the present invention is used for gene transfer and expression, and may be inserted at any position between the 5'LTR and the 3'LTR.

Examples of the heterologous promoter include an IE (immediate-early) promoter of a human cytomegalovirus (HCMV), a human GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) promoter, a human EF1-alpha (elongation factor 1-alpha) promoter, A human beta-actin promoter can be used.

The recombinant retroviral vector of the present invention can be produced by a known method for producing a recombinant viral vector and includes a multicloning site for facilitating the introduction of a foreign gene between the 5'LTR and the 3'LTR , And a restriction site for linearization.

Further, the present invention relates to (1) the MLV-derived recombinant retrovirus vector; And (2) an expression cassette comprising a selectable marker gene, a heterologous promoter operably linked thereto, and a polyadenylation signal.

At this time, the neomycin resistance gene, puromycin resistance gene, hygromycin resistance gene, O (6) -methylguanine-DNA methyltransferase (MGMT) gene, And the? -LNGFR (p75 low affinity nerve growth factor receptor) gene from which the transfer domain is removed.

More preferably, the recombinant retrovirus vector of the present invention is a recombinant plasmid pI-LND-n having the gene map of Fig.

The recombinant retroviral vector or recombinant plasmid of the present invention can be prepared by transfecting it with a suitable host cell by methods known to those skilled in the art.

In one embodiment of the present invention, the ability of the recombinant retrovirus vector of the present invention inserted into the target cell chromosome to activate the peripheral gene was compared with the conventional vector.

As a result, it was confirmed that by removing only the enhancer portion of U3 in the LTR of the retroviral vector, it is possible to lower the possibility of activation of peripheral gene expression, which is a risk of concern after insertion of the retroviral vector into the host chromosome (see Tables 1 and 2) 2).

On the basis of the above facts, in another embodiment of the present invention, various vectors having different deletion sites of 3'LTR of U3 were prepared, and then they were ligated to MLV gag - pol , Gibbon-ape leukemia virus 293T cells were transfected with a plasmid expressing the env gene of the gibbon ape leukemia virus and the viral titer was transfected into the fibrosarcoma cell line to obtain the NCBI registration (PI-D1-SV-Neo) in which nucleotide numbers 7847 to 8113 are deleted (pI-D2-SV-Neo) in which 7847 to 8161 are deleted and 7847 to 8208 are deleted (PI-D3-SV-Neo) was similar to that of the wild-type U3 control (Table 2 and Table 3).

In another embodiment of the present invention, the pI-D2-SV-Neo vector was transformed into a form suitable for gene therapy and transfected into 293T cell and PG13 cell line to confirm viral titer. As a result, And the virus titer was obtained.

Accordingly, the present invention provides a recombinant retrovirus producing cell line comprising the MLV-derived recombinant retroviral vector or the recombinant plasmid; A method of culturing the production cell line to produce recombinant retrovirus; And a cell transfected with the recombinant retrovirus produced from the production cell line.

The present invention also relates to a heterologous gene and a heterologous promoter operably linked thereto; And a recombinant retrovirus (wherein x is 7800 to 7900 and y is 8100 to 8260) comprising the genome of an MLV-derived recombinant retrovirus deleted from the nucleotide numbers x to y of the U3 portion of the LTR of MLV As an active ingredient, a pharmaceutical composition for gene therapy.

The composition may further comprise at least one pharmaceutically acceptable carrier. As the carrier, the excipient or diluent may be selected from the group consisting of saline, buffered saline, dextrose, water, glycerol and cell culture medium However, the present invention is not limited thereto. The composition may be administered orally or parenterally.

Preferably, ex vivo gene therapy can be performed using cells transfected with the recombinant retroviral vectors of the present invention, as in conventional gene therapy using retroviral vectors. The exact dosage is preferably varied depending on the condition of the patient, the type of disease, and the drug being used, and such dosage is determined through preclinical and clinical phase 1.

As described above, the recombinant MLV-derived recombinant retroviral vector of the present invention has a low possibility of unnecessarily activating a peripheral gene of a target cell, thus being safe, having a high virus titer and excellent in gene transfer efficiency, have.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example 1: Preparation of U3 deletion vector

In order to develop a retroviral vector with a structure that minimizes the possibility of activation of the surrounding gene after insertion into the host chromosome, a vector obtained by removing the enhancer portion of U3 was prepared. Respectively.

The retroviral vector is characterized by random insertion into the chromosome of the host cell. Therefore, it is impossible to compare the activation effect on the surrounding genes under the same conditions when the general retroviral vector is used. For comparison under the same conditions, a plasmid was added with a retroviral vector and a marker gene capable of quantifying the promoter and gene expression level of the gene that could be affected therefrom. Using this method, a retroviral vector of various structures can be inserted in the same position for a specific promoter and gene. Therefore, it is possible to quantitatively compare the effect of the retroviral vector structure on a specific promoter through the expression amount of a specific gene.

(Step 1) Preparation of pNRT-CAT plasmid

The retroviral vector and its surrounding sequences were constructed similar to those of leukemia patients who developed X-SCID gene therapy reported in 2003 (Kohn et al., Nat Rev Cancer 3: 477-88, 2003). That is, the promoter upstream sequence and the promoter portion and the 5'UTR (untranslated region) of the human LMO2 gene were sequentially placed at 5.0 kb downstream of the retrovirus vector portion. The 5'UTR of 0.43 kb size is known to contain important sequences in LMO2 transcription (Crable et al., Blood 101: 4757-64, 2003). The GFP-IRES-Neo expression cassette was inserted into the retroviral sequence to enable selection of the plasmid-transferred cells. The CAT gene was inserted downstream of the 5'UTR of LMO2 to quantitatively analyze the possibility of activation of the retroviral vector of each structure through the expression amount of CAT gene.

(Step 2) Preparation of pUC-LMO2P

First, in order to clone the promoter upstream portion, the promoter and the 5'UTR portion of the LMO2 gene from the genomic DNA of K562 cells (ATCC CCL-243), primer pairs of SEQ ID NOS: 1 and 2 were used PCR was performed. At this time, the PCR was carried out in the same manner as in Example 1, except that 1 μl of template DNA (100 ng of genomic DNA of K562 cells), 1 μl of each 10 pmol / μl primer, 5 μl of 10 mM dNTP, 3.5 units of an Expand High Fidelity enzyme (Gibco BRL, USA) And the reaction solution was denatured at 94 ° C for 5 minutes and then reacted at 94 ° C for 1 minute, at 50 ° C for 1 minute, and at 72 ° C for 1 minute and 30 seconds to 30 And then finally amplified at 72 ° C for 10 minutes.

The amplified PCR product was subcloned into a pGemT easy plasmid (Promega, WI, USA) to prepare a plasmid pGemT-LMO2P, and a plasmid prepared by sequencing was identified. A pUC-LMO2P plasmid was prepared by inserting the gene fragment obtained by treating the above prepared pGemT-LMO2P plasmid with SalI / Smal into SalI / SmaI sites of pUC18 (Invitrogen, USA).

(Step 3) Preparation of pNRT-CAT plasmid

To clone the CAT gene and the poly A signal region from the pCN plasmid (Lee et al ., Biochem Biophys Res Commun 272: 230-35, 2000), the primer pairs of SEQ ID NOS: 3 and 4 were used PCR was performed. At this time, the PCR was carried out using 1 μl of template DNA (100 ng of pCN plasmid), 1 μl of each 10 pmol / μl primer, 5 μl of 10 mM dNTP, 3.5 units of Expand High Fidelity enzyme (Gibco BRL, USA) The reaction mixture was denatured at 94 ° C for 5 minutes and then repeated 30 times at 94 ° C for 1 minute, 50 ° C for 1 minute, and 72 ° C for 1 minute 30 seconds, And finally amplified at 72 ° C for 10 minutes.

The amplified PCR product was inserted into a pGemT easy plasmid to prepare a pGemT-CATpA plasmid, and the plasmid prepared by the nucleotide sequence analysis was confirmed. A pNRT-CAT plasmid was prepared by inserting the gene fragment obtained by treating the pGemT-CATpA plasmid prepared above with SmaI into the SmaI site located downstream of the LMO2 5'UTR of the pUC-LMO2P plasmid prepared above.

(Step 4) Preparation of plasmids with different deletion sites

Plasmids containing retrovirus sequences with various deletion LTRs were prepared by inserting retrovirus sequences into HindIII / SalI restriction enzyme sites located at the most upstream of the pNRT-CAT plasmid prepared in step 3 (FIG. 1).

First, the CAT gene of the pMIN-CAT retrovirus vector (Yu et al., Gene Ther 7: 797-804, 2000) was removed by treatment with MluI / BglII and then pMT-GFP retrovirus vector (Hong et al., J Gene Med 6: 724-33, 2004) was treated with MluI / BglII to obtain a pMIN-GFP retroviral vector. A pMT-CAT plasmid was prepared by inserting a gene fragment containing the entire retrovirus vector sequence obtained by treating the pMIN-GFP retrovirus vector with HindIII / SalI into the HindIII / SalI site of pNRT-CAT.

Plasmids other than pNRT-CAT and pMT-CAT were prepared by replacing LTRs in which the U3 portion of the LTRs of MLV was removed. The U3 deletion LTR is a form in which the nucleotide sequence from the 32nd nucleotide to the 298th nucleotide sequence is removed from the U3 portion of the LTR (the nucleotide sequence of nucleotide No. 7847-8113 is deleted, based on NCBI registration number J02255). The removed portion has controlled the cis-acting regulatory sequences, and the (cis -acting regulatory sequence) of YY1, NFAT, ELP, and DR (direct repeats) are present, it affects the transcription from the LTR (Wahlers etc., Mol Ther 6: 313-20, 2002).

In order to prepare a plasmid having a U3 deletion LTR, a pMT retrovirus vector having a wild type LTR derived from MLV (Yu et al., Gene Ther 7: 797-804, 2000; U.S. Patent No. 6,451,595) was treated with XhoI / The 3'LTR fragment was inserted into the XhoI / SalI site of the pUC plasmid to prepare the pUC-3LTR plasmid. pUC-3LTR was treated with NheI / XbaI to separate the U3 portion of the LTR, and the remaining plasmid portion was self-ligation to prepare pUC-d3LTR.

On the other hand, the pMIN-GFP vector used in the production of the pMT-CAT plasmid was treated with XhoI / SalI to remove the 3'LTR portion, and then the pUC-d3LTR prepared above was treated with XhoI / And the pMIN-GFP-d3LTR vector was prepared. This vector was further treated with NheI / XbaI to remove the U3 portion of the 5'-LTR portion, and only the vector portion was self-ligated, resulting in a pMIN-GFP-dLTR plasmid in which both the 5 'and 3'LTR were deleted . Then, the pMIN-GFP-dLTR plasmid was treated with HindIII / SalI to isolate only the retroviral vector portion and inserted into the HindIII / SalI site of pNRT-CAT to prepare pI-D-CAT.

The pMIN-GFP-dLTR plasmid prepared in the pI-D-CAT production process was further treated with XhoI / SalI, and then the 3'LTR gene fragment obtained by treating the pMIN-GFP vector with XhoI / SalI was inserted to obtain pMIN -GFP-d5LTR vector. Then, the pMIN-GFP-d5LTR vector was treated with HindIII / SalI to isolate only the retroviral vector portion and inserted into the HindIII / SalI site of pNRT-CAT to prepare p5'-I-D-CAT.

The p3'-I-D-CAT plasmid was prepared by treating the pMIN-GFP-d3LTR vector with HindIII / SalI to isolate only the retroviral vector portion and inserting it into the HindIII / SalI site of pNRT-CAT.

The pC-D-CAT plasmid has the HCMV IE promoter inserted at the MluI / BamHI site of the pMIN-GFP-dLTR vector. The IE promoter gene fragment of HCMV obtained by treating the pCN plasmid (Lee et al ., Biochem Biophys Res Commun 272: 230-35, 2000) with MluI / BamHI restriction enzyme was inserted into the pMIN-GFP-dLTR vector to construct pMIN-C-GFP-dLTR Vector. This vector was treated with HindIII / SalI to isolate only the retroviral vector portion and inserted into the HindIII / SalI site of pNRT-CAT to prepare pC-D-CAT.

≪ Test Example 1 > Measurement of expression rate of peripheral gene CAT [

10 μg of each of the plasmids prepared above was linearized using NdeI restriction enzyme and then introduced into K562 cells (ATCC CCL-243) by electroporation (BIORAD, USA). From day 1, G418 sulphate (Gibco- BRL, NY, USA), and only the cells transformed with the plasmid containing NEO were selected. Approximately two weeks later, the selected cell lines were harvested for each plasmid and the CAT gene expression was compared as follows (Gorman et al., Mol Cell Biol 2: 1044-51, 1982).

Selected cell populations with each plasmid were harvested, washed with PBS buffer, and suspended in 0.25 M Tris-HCl (pH 7.5). The cells were lysed by 5 consecutive freeze-thaw cycles and then incubated at 65 ° C for 7 min to inactivate enzymes other than CAT protein. The CAT gene expression level was then measured by CAT assay Respectively. Each 25 μg of the cell protein was reacted with 2 μl of acetyl-CoA (40 mM) and 1 μl of C ¹⁴ -chloramphenicol (30 minutes to 1 hour), and then 400 μl of ethyl- Respectively. The recovered material was concentrated by centrifugation in vacuo and then dissolved in the final 15 μl of ethyl acetate and applied to thin-layer chromatography (TLC) plate to obtain a developing solution (95% chloroform, 5 % Methanol) for 30 minutes. After development, the ratio of acetylated chloramphenicol to the total amount of chloramphenicol was quantified using a phosphoimager (FUJIX BAS 1000). At this time, the results of each test group were calculated by converting the results of pNRT-CAT to 1, and in all cases, three or more tests were performed and the mean and standard deviation were obtained.

As shown in Table 1, in the case of pMT-CAT having wild-type LTRs in both LTRs, it was confirmed that the activity of CAT gene was increased about 7 times as compared with pNRT-CAT having no retrovirus vector. This means that it is possible to induce the gene expression of the promoter of downstream cells even when the LTR is present about 5.0 kb upstream on the chromosome.

pI-D-CAT exhibits a very low CAT expression rate, meaning that retroviral vectors with deletions of the U3 of the 5 'and 3' LTRs have extremely low effects on downstream genes.

In order to actually use the retrovirus vector using the deleted LTR for gene transfer and expression, an internal promoter that promotes the expression of the target gene should be separately used. As shown in Table 1, pC-D-CAT in which the HCMV promoter was inserted showed low CAT gene expression similar to that of pI-D-CAT in which the HCMV promoter was not inserted, It means that the use of the promoter does not greatly affect the activation of the peripheral gene.

In addition, depending on the position of the LTR from which U3 was deleted, the effect of activating the LMO2 promoter was significantly different. CAT expression in the case of deletion of U3 of 5'LTR (p5'-ID-CAT) is increased about 11-fold compared to that of pNRT-CAT, but when the U3 of 3'LTR is deleted (p3'-ID-CAT) CAT expression was only about 1.6 times. This fact implies that the effect on the promoter activation of the LTR (3 'LTR in this experiment) adjacent to the downstream promoter is greater than that of the LTR (5' LTR) remote from the promoter.

≪ Test Example 2 > Measurement of expression rate of peripheral gene CAT - Northern blotting analysis

The degree of CAT expression by the retrovirus vector prepared in the above Example was compared using Northern blotting. RNA was isolated from the K562 cell lines transformed with each plasmid by using guanidine thiocyanate-cesium chloride method, and 10 의 of RNA was added to 1% formaldehyde-agarose gel -agarose gel. The agarose gel was transferred to a nitrocellulose (NC) membrane, and fusion reaction was induced by using the entire base sequence of the ³² P-labeled CAT gene as a probe. At this time, the difference in the amount of RNA developed on the agarose gel was confirmed by expanding the amount of beta-actin RNA of the cells together in the same nitrocellulose membrane. The CAT gene probe was obtained by treating the pCN-CAT plasmid with the BamHI fragment, and the beta-actin gene probe was obtained by using EcoRI-treated gene fragment of Actin cDNA. The results are shown in FIG.

The arrows in Fig. 2 represent the relative positions of the 28S and 18S rRNAs of the cells, respectively, which can be used as markers indicating 5.02 kb and 1.86 kb positions on 1% agarose gel.

As a result of the experiment, similar to the results of the CAT analysis, when the wild-type LTR was present on both sides of the retroviral vector (column 2: pMT-CAT) and only on the 3 'side (column 4: p5'-ID- CAT), a relatively strong signal of CAT mRNA was detected compared to the other. Considering the positions of 28S and 18S rRNA, the following mRNA among the two kinds of RNA shown in the result is expressed from the LMO2 promoter, and it can be judged that the above mRNA is expressed from the 3'LTR. These results indicate that LMO2 gene is activated by retroviral vector with LTR at the mRNA level.

Example 2 Preparation of Retroviral Vectors with Different U3 Removal Sites

To determine the optimum site of LTR for obtaining safety and high viral titers, a series of retroviral vectors with different LTR removal sites were prepared as follows (FIG. 3).

The wild-type 3'LTR portion of MLV was amplified by PCR using a primer pair described as pMT (US Patent No. 6,451,595) and described as SEQ ID NO: 5 (SCV3LB) and 6 (SCV3LRI). The amplified PCR product was inserted into the NheI / SalI site of the pGEM T easy vector to construct the pGEM T easy-3'LTR plasmid. Then, the NheI-XbaI fragment of the pGEM T easy-3'LTR was removed and self-ligation was performed to make pGEM T easy-3'd LTR.

The PCR product amplified with the primer pair described in SEQ ID NO: 5 (SCV3LB) and SEQ ID NO: 7 (3LTR-1) was inserted into the NheI / SalI site of pGEM T easy vector using pMT as a template, dLTR1 plasmid. The NheI-SalI fragment of this plasmid was inserted into the NheI-SalI site of the pGEM T easy-3'LTR to prepare pPreSIN2.

A pGEM T easy-3'd LTR2 plasmid was prepared by inserting the PCR product amplified with the primer pair described in SEQ ID NO: 5 (SCV3LB) and SEQ ID NO: 8 (3LTR-2) into the NheI / SalI site of the pGEM T easy vector. The NheI-SalI fragment of this plasmid was inserted into the NheI-SalI site of pGEM T easy-3'LTR to prepare pPreSIN3.

A pGEM T easy-3'd LTR3 plasmid was prepared by inserting the PCR product amplified with the primer pair described in SEQ ID NO: 5 (SCV3LB) and SEQ ID NO: 9 (3LTR-3) into the NheI / SalI site of the pGEM T easy vector. The NheI-SalI fragment of this plasmid was inserted into the NheI-SalI site of the pGEM T easy-3'LTR to prepare pPreSIN4.

The pGEM T easy-3'd LTR4 plasmid was prepared by inserting the PCR product amplified with the primer pair described in SEQ ID NO: 5 (SCV3LB) and SEQ ID NO: 10 (3LTR-4) into the NheI / SalI site of the pGEM T easy vector. The NheI-SalI fragment of this plasmid was inserted into the NheI-SalI site of the pGEM T easy-3'LTR to prepare pPreSIN5 (Fig. 3).

Subsequently, a BamHI-SalI fragment was obtained from the pGEM T easy-3'd LTR plasmid prepared above, and then inserted into a retrovirus vector pMT treated with BamHI-SalI to prepare a retrovirus vector pI-D1, and pPreSIN2, pPreSIN3, pPreSIN4 And the BamHI-SalI fragment of pPreSIN5 were respectively inserted into the BamHI-SalI site of the retroviral vector pMT to construct retroviral vectors pI-D2, pI-D3, pI-D4 and pI-D5 (FIG.

≪ Test Example 3 > Comparison of virus titers after pSV-Neo cassette insertion

To compare the viral titers of the series of retrovirus vectors prepared in Example 2, pSV-Neo cassettes were inserted into each vector as described below, and the number of colonies resistant to neomycin was measured .

The pSV-Neo cassette was obtained by treating the pDON-AI vector (Takara Bio, Otsu, Japan) with BamHI-XhoI and obtaining the MT vector and pI-D1, pI-D2, pI- D1-SV-Neo (vector in which nucleotide numbers 7847 to 8113 are deleted based on NCBI registration number J02255), pI-D2-SV (SEQ ID NO: -Noo (vector in which nucleotide numbers 7847 to 8161 are deleted based on NCBI registration number J02255), pI-D3-SV-Neo (vector in which nucleotide numbers 7847 to 8208 are deleted based on NCBI registration number J02255) , pI-D4-SV-Neo (vector in which nucleotide numbers 7847 to 8244 were deleted based on NCBI registration number J02255), and pI-D5-SV-Neo vector (based on NCBI registration number J02255, nucleotide number 7847 To 8264 were deleted).

The prepared retroviral vector DNA was amplified by PCR using pVM-gp and pVM-GeR plasmid DNA (Yu et al., Gene (2002)) expressing the env gene of the gag - pol of MLV, gibbon ape leukemia virus Ther 10: 706-11, 2003) infection (transfection) on 293T cells transfected, 37 ℃, and with a CO ₂ incubator (carbon dioxide partial pressure of FBS (Fetal bovine serum 10% eseo 5%); Gibco-BRL, Grand Island, After incubation for 48 hours in DMEM (CAMBREX, 12-604F) supplemented with DMEM (NY, USA, Cat. 26140), cell culture was filtered with a 0.45 ㎛ filter to obtain virus-free virus solution. 1 × 10 ⁵ HT1080 cells (human fibrosarcoma cell line; ATCC CCL-121) were prepared in a 60 mm dish, and the virus solution was continuously transfused the following day, and the cells were cultured for 1 day. The next day, transfected HT1080 cells were transferred to a 100 mm dish, and G-418 sulfate (Gibco, NY, USA) was added to give 1 mg / ml and cultured for about 2 weeks. After culturing, the medium was removed, and the cells were stained with crystal violet to measure the number of colonies, and the relative titer of virus was determined and shown in Table 2 below.

As a result, as shown in Table 2, I-D1 and I-D2 produced viruses at a level similar to the MT vector having the wild-type LTR.

<Test Example 4> Comparison of virus titers after GFP gene insertion

To compare the effect of the foreign gene on the virus titer when the foreign gene was further inserted into the series of retrovirus vectors prepared in Test Example 3, the reporter gene GFP was inserted into each vector and the virus titer was compared Respectively.

PI-D2-SV-Neo and pI-D3 prepared in Example 2 were obtained after obtaining BamHI-BglII fragment of pGem T easy-eGFP prepared in step 2 of Example 3 below. -GV-Neo, pI-D3-GFP-SV-Neo, pI-D3-GFP-SV-Neo and pI-D4-SV- -D4-GFP-SV-Neo vector.

The prepared vector DNA was transfected into 293T cells together with pVM-gp and pVM-AE plasmid DNA (Yu et al., Gene Ther 10: 706-11, 2003) expressing the gag-pol and env genes of MLV, respectively, , And cultured in a CO ₂ incubator (carbon dioxide partial pressure 5%) for 48 hours The cell culture was filtered with a 0.45 mu m filter to obtain a virus solution from which cells were removed. 1 × 10 ⁵ HT1080 cells were prepared in a 60 mm dish, and the following day, 1 μl of the virus solution was added and transfected, followed by culturing for 1 day. The next day, transfected HT1080 cells were transferred to a 100 mm dish and G-418 sulfate was added to give 1 mg / ml and cultured for about 10 days. After the culture, the medium was removed and the cells were stained with crystal violet to measure the number of colonies, and the relative titer of virus was determined and shown in Table 3 below.

In Test Example 3, the viruses I-D1 and I-D2 produced viruses at levels similar to those of the MT vector having the wild-type LTRs. In the case of I-D3 and I-D4, the titer of the virus was reduced to less than half. However, in the vector into which the reporter gene GFP was inserted, I-D1-, I-D2- and I-D3-GFP-SV-Neo produced viruses at similar levels as shown in Table 3, Only in the case of -SV-Neo, the virus titer decreased to less than half.

From the results of Test Examples 3 and 4, it can be seen that I-D1, I-D2 and I-D3 produce viruses at similar levels. However, since I-D2 and I-D3 contain fewer U3 sites than I-D1, the possibility of peripheral gene activation by retroviral vector insertion is relatively low, so I-D2 or I-D3 It is preferable to use a retrovirus in the form of a virus.

&Lt; Example 3 > Development of retroviral vector in a form suitable for gene therapy

(Step 1) Preparation of retroviral vector pI-LND-n

Based on the retrovirus vector pI-D2 prepared in Example 2, a retrovirus vector maximizing convenience was developed.

First, a new multi-cloning site (MCS) was introduced to easily clone the desired therapeutic gene into the retroviral vector pI-D2. The 52 bp long MCS was prepared by polymerase-free reaction using a pair of synthetic oligonucleotide primers of SEQ ID NO: 11 (NEWMCSF) and 12 (NEWMCSR).

As the PCR reaction conditions, 1 μl of each 10 pmol / μl primer, 10 μl of 10 mM dNTP, 3.5 units of Expand High Fidelity enzyme (Gibco BRL, USA) and 10 μl of enzyme buffer solution were mixed to give a total of 100 Mu] l of the reaction solution, and this reaction was performed once at 94 [deg.] C for 1 minute, at 55 [deg.] C for 1 minute, and at 72 [deg.] C for 1 minute 30 seconds. The amplified fragments had restriction sites of MluI, PmeI, SacII, EcoRI, BamHI, DraIII, StuI, BglII, and XhoI and this fragment was inserted into pGEM T easy vector to produce pGEM T easy-nMCS plasmid . After confirming the sequence was confirmed by sequencing, the MluI-XhoI fragment was inserted into the MluI-XhoI site of pI-D2 to prepare pI-ND.

On the other hand, a retroviral vector-producing cell line having a wild-type LTR can be produced using both methods of transduction or transfection. To produce a retrovirus vector producing cell line lacking the U3 region Retroviral vectors in the form of plasmid DNA must be introduced into the genome of the packed cell line by the transfection method. This is because, in the case of a retrovirus vector in which the U3 region has been deleted, once the U3 of both LTRs is deleted after transfection, the genomic RNA of the virus can no longer be generated. In addition, when a production cell line is to be produced by the transfection method, it is necessary to linearize the plasmid DNA-type retroviral vector in order to insert it into the genome while maintaining the integrity of the DNA structure. In this Example, restriction enzyme sites for linearization were introduced as follows.

Two pieces L1 and L2 containing the restriction site for linearization were inserted at the front of the 5'LTR and 200bp behind the 3'LTR, respectively. The L1 fragment was prepared by performing a polymerase reaction under the same conditions as above using a pair of synthetic oligonucleotide primers of SEQ ID NO: 13 (L1F) and 14 (L1R) without template.

The amplified fragments had restriction sites of SapI, PmlI, BclI, SwaI, BstBI, and SapI and inserted into the pGEM T easy vector to construct the pGEM T easy-L1 plasmid.

L2 fragment was prepared by PCR amplification using a pair of synthetic oligonucleotide primers of SEQ ID NO: 15 (L2F) and 16 (L2R) using pUC18 (Promega, Wis., USA) as a template.

As the PCR reaction conditions, 1 μl of template DNA, 1 μl of 10 pmol / μl primer, 10 μl of 10 mM dNTP, 3.5 units of Expand High Fidelity enzyme (Gibco BRL, USA) and 10 μl of enzyme buffer solution To prepare a total reaction solution of 100 μl. The reaction solution was repeated 30 times at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 1 minute 30 seconds. The amplified fragments had restriction sites of StuI, BstEII, SfiI, PmlI, BclI, SwaI, and SspI and inserted into the pGEM T easy vector to construct the pGEM T easy-L2 plasmid. After confirming sequencing of L1 and L2 sequences, the SapI fragment of pGEM T easy-L1 was inserted at the SapI site of pI-ND and the StuI-SspI fragment of pGEM T easy-L2 was inserted at the SspI site of pI-ND To prepare pI-LND.

In order to produce a virus-producing cell line having high activity, it is necessary to select only cells containing retrovirus vector DNA. A selectable marker gene is often used for this purpose. When a selectable marker gene is contained in a retroviral vector, when a vector or a vector containing a vector is injected into the organism, an immune response to the selectable marker gene There is a possibility that the cells will disappear without being maintained for a long time. Therefore, in order to use a vector for gene therapy, it is preferable that the vector does not contain a selectable marker gene.

In this example, the selection gene was introduced into the outside of the retroviral vector genome in order to facilitate the selection of the producing cell line of the retrovirus vector. The neomycin resistance gene was used as a selection gene. To express the neomycin resistance gene, an expression cassette was prepared in the following manner.

First, the human beta-actin promoter was cloned by PCR using the genomic DNA obtained from the K562 cell line as a template and using a synthetic oligonucleotide primer pair of SEQ ID NO: 17 (BApF) and 18 (BApR).

The PCR reaction conditions are the same as those used in the production of the L2 fragment in the above. The amplified fragments were inserted into pGEM T easy vector to prepare pGEM T easy-pBA plasmid. Sequencing confirmed the correct sequence.

The fragment to which the neomycin resistance gene and the polyadenylation signal (pA) were connected was prepared as follows.

The neomycin resistance gene was amplified by PCR using a pair of synthetic oligonucleotide primers of SEQ ID NO: 19 (Neo 5) and 20 (Neo 3) using pcDNA 3.1 (Invitrogen, CA, USA) as a template.

The PCR reaction conditions were the same as those used in the preparation of the L2 fragment in Example 3. The amplified fragments were inserted into pGEM T easy vector to construct pGEM T easy-Neo plasmid. Sequencing confirmed the correct sequence.

The polyadenylation signal (pA) was amplified by PCR using a pair of synthetic oligonucleotide primers of SEQ ID NO: 21 (SVpA 5) and 22 (SVpA 3) using pTet-On (Clontech, TAKARA bio, Respectively.

The PCR reaction conditions are the same as above. The amplified fragment was inserted into the pGEM T easy vector to construct a pGEM T easy-pA plasmid. Sequencing confirmed the correct sequence.

pGEMT easy-Neo-pA was prepared by inserting the XhoI-NdeI fragment of pGEM T easy-pA into the XhoI-NdeI site of pGEM T easy-Neo.

pGEM T easy-pBA-Neo-pA was prepared by inserting a BglII-NdeI fragment of pGEM T easy-Neo-pA into the BamHI-NdeI site of pGEM T easy-pBA. Then, the end of the MluI-EcoRI fragment obtained from pGEM T easy-pBA-Neo-pA was smoothed with Klenow enzyme and inserted into the SspI site of retrovirus vector pI-LND to obtain pI-LND-n vector (Fig. 5).

(Step 2) Preparation of retroviral vector pC-LND-GFP-n

Since the pI-LND-n vector prepared in (step 1) does not have a promoter capable of expressing the target gene, an internal promoter is separately introduced to express the target gene. An MluI-BamHI fragment containing the IE (immediate-early) promoter of human cytomegalovirus (HCMV) was obtained from pCN (Lee et al ., Biochem Biophys Res Commun 272: 230-35, 2000) -LND-n at the MluI-BamHI site to prepare pC-LND-n.

Enhanced green fluorescence protein (eGFP) was inserted as a reporter gene so that the gene expression ability of the pC-LND-n vector prepared above could be confirmed. The eGFP gene was amplified by PCR using a pair of synthetic oligonucleotide primers of SEQ ID NO: 23 (eGFP5) and 24 (eGFP3) from pIRES2-EGFP (CLONTECH Laboratory, Palo Alto, CA, USA, Cat. # 6029-1) . The PCR reaction conditions are the same as those used in the production of the L2 fragment in the above.

The pGEM T easy-eGFP plasmid was prepared by inserting the amplified PCR product into pGEM T easy vector. A BamHI-BglII fragment of this plasmid was inserted at the BamHI site of the retroviral vectors. pC-LND-GFP-n was prepared by inserting a BamHI-BglII fragment of pGEM T easy-eGFP into the BamHI site of pC-LND-n (FIG.

<Test Example 5> Confirmation of performance of C-LND-GFP-n in 293T cells

To examine the performance of the retrovirus vector pC-LND-GFP-n prepared in (Step 2) of Example 3, the vector DNA was ligated with pVM-gp and pVM-gp expressing the gag-pol and env genes of MLV, 293T cells were transfected with AE plasmid DNA (Yu et al., Gene Ther 10: 706-11, 2003) to produce viruses. After 8 hours from transfection, the medium (DMEM medium supplemented with 10% FBS) was changed and cultured for 48 hours in a CO ₂ incubator (carbon dioxide partial pressure 5%) at 37 ° C. The cells were filtered to obtain a virus solution from which cells were removed. C-LND-GFP-n retrovirus of this solution was transfected into HT1080 cell line as follows.

1 x 10 &lt; ⁵ &gt; HT1080 cells were plated in a 60 mm dish one day before transfection. Cells were harvested and analyzed by FACS analysis (FIG. 6B). Cells were harvested after adding 500 μl of the viral solution obtained above with polybrene (final 8 μg / ml) for 48 hours.

FACS analysis was performed as follows: Cells were harvested and cells were washed with PBS (phasphate-buffered saline) containing 0.1% sodium azide. Then, the ratio of fluorescence-emitting cells and fluorescence intensity were analyzed using a FACSort instrument (Becton Dickinson). CellQuest (Becton Dickinson) was used for data acquisition and analysis. As control, HT1080 cells that did not transfer the virus, that is, did not transduce, were used.

The results showed that the retroviral vector pC-LND-GFP-n induces a high level of gene expression (average fluorescence intensity: 1265) and that it can produce high reverse transcriptase viruses (83% of the cells express GFP) 7).

<Test Example 6> Confirmation of the performance of pC-LND-GFP-n in the PG13-producing cell line

In actual gene therapy, retroviral vectors from production cell lines are used rather than viruses made from 293T cells. Therefore, in order to determine whether the produced retroviral vector can be used for gene therapy, it is necessary to confirm the gene expression and the viral activity after producing the production cell line. To this end, the retrovirus vector pC-LND-GFP-n was transferred to the packaging cell line PG13 (ATCC CRL-10686) to produce PG13-C-LND-GFP-n. The overall experimental procedure is shown in Figure 8b.

First, the retroviral DNA pC-LND-GFP-n was linearized by treatment with restriction enzyme SwaI (Fig. 8a), which was observed when transfection and plasmid insertion into the chromosome of the cell line This is because the plasmid structure may not be properly maintained. The linearized retroviral vector DNA was transfected using the electroporation method under the following conditions.

7.5 × 10 ⁵ PG13 cells were mixed well in 0.5 ml of serum-free DMEM medium and placed in a 0.4-cm cuvette (Bio-Rad Laboratories, Hercules, Calif.). On the other hand, retroviral DNA pC-LND-GFP-n was linearized by treatment with restriction enzyme SwaI, and 10 μl (0.1 μg / μl) of the plasmid was added to the cuvette containing the cells and placed at room temperature for 5 minutes. After that, electrophoresis was carried out using Gene Pulser Xcell ( ^TM) (Bio-Rad) under conditions of 200 V and 20 ms, and 10 ml of DMEM medium supplemented with 10% FBS was added thereto. . After 24 hours, G-418 sulfate (Gibco, NY, USA) was treated with a concentration of 1 mg / ml and cultured for 2 weeks to obtain a production cell line PG13-C-LND-GFP into which retroviral vector DNA was introduced.

5 × 10 ⁶ production cell lines obtained from the above procedure were cultured for 48 hours in DMEM medium supplemented with 10% FBS at 37 ° C. in a CO ₂ incubator (carbon dioxide partial pressure 5%), and then cell culture was obtained and 0.45 μm Followed by filtration through a filter to obtain a virus solution from which cells had been removed.

The C-LND-GFP-n retrovirus obtained above was transfected into the HT1080 cell line and cultured for 48 hours. Then, the target cells were analyzed by FACS analysis. The experimental procedure was as follows: 1 x 10 ⁵ HT1080 cells were plated on a 60 mm dish one day before transfection. The cells were harvested and analyzed by FACS analysis (control was performed using viruses that did not transfer the virus, that is, HT1080 that did not transduce the virus (&lt; RTI ID = 0.0 &gt; Cells).

As a result of the analysis, it was found that the retrovirus vector was formed properly in the PG13 cell line because the 6.5% cell expresses GFP even though the virus level is lower than that of the 293T cell line (FIG. 9).

To analyze gene expression and activity in other cell lines, C-LND-GFP-n retrovirus from a production cell line was transfected into K562 cell line (ATCC CCL-243). The method of transfection into K562 is as follows: 1 × 10 ⁵ cells were placed in each well of a 6-well plate and 500 μl of virus and polybrene (final 8 μg / ml) were added. After centrifugation at 2800 rpm for 2 hours, the cells were incubated at 37 ° C for 2 hours. After the incubation, the medium was replaced with a fresh medium containing no virus. After 48 hours of incubation, cells were analyzed by FACS analysis (using K562 cells that did not transfer the virus, that is, did not transduce) as a control.

Analysis showed that effective GFP expression could be observed (12% cells expressing GFP), thus confirming once again that the retroviral vector was being properly constructed in the PG13 producing cell line (FIG. 10).

Quantitative analysis of the viral titers produced in the production cell lines through the real-time quantitative PCR method (Korean Patent No. 440852) revealed that viruses of about 5 × 10 ⁴ viral particles / ml were produced.

When a production cell line used in the present invention is subjected to a clone screening process, a high-producer clone having a production efficiency 10 times higher than that in a populated state can be obtained. Therefore, when a clone screening process is performed, ^It would be possible to obtain a cell line that produces viruses at a level of ⁵ virus particles / ml or more.

The viral vector of the present invention has low possibility of activating a peripheral gene of a target cell and is safe and can be usefully used for gene therapy because the viral titer and the expression rate of a target gene are kept high.

<110> VIROMED LIMITED <120> Retroviral vectors with improved safety <160> 25 <170> Kopatentin 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for LMO2 <400> 1 gtcgacatgc tgctttcaaa atgtctagtt 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for LMO2 <400> 2 cccgggaagc ttgaagactt gtaccccttt 30 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for CAT <400> 3 cccgggatgg agaaaaaaat cactgg 26 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for CAT <400> 4 cccgggtccc cagcatgcct gcta 24 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> SCV3LB <400> 5 ggatccctcg agcgataaaa taaaagattt tatttagtct cc 42 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> SCV3LRI <400> 6 gaatccgtcg actgaaagac ccccgctgac gg 32 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 3LTR-1 <400> 7 gctagcccct gtgccttatt tgaa 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 3LTR-2 <400> 8 gctagcttcg cgcgcttctg ctcc 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 3LTR-3 <400> 9 gctagcccca caacccctca ctcg 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 3LTR-4 <400> 10 gctagcgcgc cagtcctccg attg 24 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> NEWMCSF <400> 11 acgcgtttaa accgcggaat tcggatccac atcgtg 36 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> NEWMCSR <400> 12 ctcgagatct aggcctcacg atgtggatcc gaattc 36 <210> 13 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> L1F <400> 13 gctcttccgc tcacgtgtga tcaatttaaa tttcgaa 37 <210> 14 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> L1R <400> 14 agcggaagag cttcgaaatt taaattgatc acacgtg 37 <210> 15 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> L2F <400> 15 aggcctggtc accggccatt atggccacgt gatcatttaa atttg 45 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> L2R <400> 16 tattcgcgcg tttcggtgat gaatatt 27 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> BApF <400> 17 gtcgacatta atgccggtga gtgagcggcg cggggccaa 39 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> BApR <400> 18 ggatccggtg gcgcgtcgcg ccgctgggtt tt 32 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Neo5 <400> 19 agatctatgg gatcggccat tgaacaa 27 <210> 20 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Neo3 <400> 20 ctcgagtcag aagaactcgt caagaaggc 29 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SVpA 5 <400> 21 ctcgagatgg gatcggccat tgaacaa 27 <210> 22 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> SVpA 3 <400> 22 catatgagta atcagccata ccacattt 28 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> eGFP5 <400> 23 acgcgtggat ccatggtgag caagggcgag 30 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> eGFP3 <400> 24 ctcgagagat ctttacttgt acagctcgtc 30 <210> 25 <211> 449 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 3 ' LTR U3 region of murine leukemia virus (MLV) <400> 25 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60 atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac 120 agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180 aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg 240 ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct 300 agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt 360 tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca 420 ataaaagagc ccacaacccc tcactcggg 449

Claims (15)

Wherein the nucleotide number x to y of the U3 portion of LTR (long terminal repeat) of murine leukemia virus (MLV), represented by the nucleotide sequence of SEQ ID NO: 25, And y is 286 to 395), MLV-derived recombinant retroviral vectors. delete The method according to claim 1, x is 32, and y is 297 to 393. The method according to claim 1, Wherein the nucleotide sequences of the structural genes gag, pol and env of MLV are completely removed. The method according to claim 1, A recombinant retroviral vector, further comprising a heterologous gene and a heterologous promoter operably linked thereto. 6. The method of claim 5, Wherein the heterologous promoter is an IE (immediate-early) promoter of human cytomegalovirus. 6. The method of claim 5, Wherein the heterologous gene is a gene expressing enhanced green fluorescence protein or gp91-phox. The method according to claim 1, Wherein the recombinant retrovirus vector further comprises a multicloning site or restriction enzyme site between the two LTRs of the MLV. (1) an MLV-derived recombinant retroviral vector according to any of claims 1 and 3 to 8; And (2) an expression cassette comprising a selectable marker gene, a heterologous promoter operably linked thereto, and a polyadenylation signal. 10. The method of claim 9, The selectable marker gene is the neomycin resistance gene, the puromycin resistance gene, the hygromycin resistance gene, the O (6) -methylguanine-DNA methyltransferase (MGMT) gene, Wherein the plasmid is selected from the group consisting of p75 low affinity nerve growth factor receptor (Δ-LNGFR) gene whose domain is deleted. 10. The method of claim 9, The plasmid pI-LND-n having the gene map of Fig. 5 is a recombinant plasmid. (1) an MLV-derived recombinant retroviral vector according to any of claims 1 and 3 to 8; or (2) an MLV-derived recombinant retroviral vector of any one of claims 1 and 3 to 8, and an expression cassette comprising a selectable marker gene, a heterologous promoter operably linked thereto, and a polyadenylation signal The recombinant plasmid A recombinant retrovirus producing cell line. A method for producing a recombinant retrovirus by culturing the production cell line of claim 12. 12. A cell transfected with a recombinant retrovirus produced from the production cell line of claim 12. A heterologous gene and a heterologous promoter operably linked thereto; And a recombinant retrovirus comprising the genome of an MLV-derived recombinant retrovirus deleted from the nucleotide numbers x to y of the U3 portion of the LTR of MLV represented by the nucleotide sequence of SEQ ID NO: 25 (wherein x is 1 to 85 , y is from 286 to 395).

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