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Vector for expressing two foreign genes

A technology for exogenous genes and vectors, which can be used in vectors, introduction of foreign genetic material using vectors, gene therapy, etc., and can solve problems such as unsatisfactory vector expression performance and promoter interference.

Inactive Publication Date: 2002-08-14
DNAVEC RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression performance of these vectors is not satisfactory
[0004] For example, vectors containing multiple promoters may have expression efficiency issues when expressing from only one of the promoters due to interference between the promoters
On the other hand, the problem with vectors containing a combination of a promoter and an IRES sequence is that when the gene is expressed from the 3' end of the IRES, the expression level is only 1 / 5 to 1 / 10 of that expressed from the 5' end of the IRES

Method used

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  • Vector for expressing two foreign genes
  • Vector for expressing two foreign genes
  • Vector for expressing two foreign genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] [Example 1] Production of SIVagm vector and identification of its performance

[0115] A new lentiviral vector was prepared using the monkey-derived non-pathogenic immunodeficiency virus clone SIVagmTY01 as follows. figure 1 A schematic of the vector system is shown.

[0116] SIVagmTY01 containing a non-pathogenic immunodeficiency virus clone derived from African green monkeys was used in the preparation of the vector system. Hereinafter, all nucleotide numbers are indicated with the transcription initiation site of the viral RNA as +1. As a plasmid, pSA212 (Journal of Virology, Vol. 64, pp. 307-312, 1990) into which SIVagmTY01 had been inserted was used. All ligation reactions were then performed using Ligation High (Toyobo) according to the attached instructions.

[0117] a. Generation of packaging carrier

[0118]First, use pSA212 as a template and use primers 1F (SEQ ID NO: 1) and 1R (SEQ ID NO: 2) to obtain the region (5337-5770) corresponding to the region (5...

Embodiment 2

[0127] [Example 2] Modification of 5'LTR

[0128] The transcriptional activity of the 5'LTR of lentiviruses generally depends on the presence of the virus-derived Tat protein. Therefore, in order to eliminate Tat dependence and to increase the vector titer by replacing it with a promoter sequence with strong transcriptional activity, a SIVagm gene transfer vector was generated, in which the U3 region as the 5'LTR promoter sequence was promoted by another The subsequence replaces ( figure 2 ).

[0129] Replacement of the 5'LTR with a chimeric promoter was achieved as follows. Using a series of primers 9-1F to 3F (SEQ ID Nos: 45-47) and primer 9R (SEQ ID NO: 48) and using pSA212 as a template to PCR amplify the region containing the TATA box downstream to the gag region in the 5' LTR Fragment of the region (9039-9170+1-982). Then, use primers 10-1F (SEQ ID NO: 49) and 10-1R (SEQ ID NO: 50) and use pCI as template respectively; use primers 10-2F (SEQ ID NO: 51) and 10-2R (SE...

Embodiment 3

[0130] [Example 3] Modification of 3'LTR

[0131] In lentiviral vectors, the promoter sequence contained in the 3'LTR is integrated as a U3 region into the U3 promoter region of the 5'LTR when the target cell is reverse transcribed. It was found that the U3 region contained in the 3'LTR region of the gene transfer vector became the U3 promoter of the 5'LTR involved in gene expression in the target cell genome ( image 3 ). Therefore, a SIVagm gene transfer vector was prepared in which the U3 region of the 3'LTR was replaced with other promoter sequences, and the evaluation of the other promoter sequences could determine whether the promoters involved in gene expression in target cells were It can also be replaced by other promoters than U3 sequences ( image 3 ). In addition, the SIVagm gene transfer vector with the U3 region of the 3'LTR deleted was prepared, and the evaluation of the vector could determine whether the promoter sequence on the 5'LTR in the target cell coul...

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Abstract

A vector expressing two foreign genes by using RRE sequence and controlling the ratio of the expression doses of these genes owing to the modification is provided. This vector, which can be provided as a lentivirus vector based on SIVagm, is constructed by modifying a virus-origin expression regulatory sequence into another expression regulatory sequence so as to eliminate the dependency on the virus-origin protein. Although this vector has a packaging signal, it has been modified so that the risk of the occurrence of wild strains due to gene recombination is lowered and no virus structural protein is expressed. This vector is highly useful as a gene therapeutic vector with a need for transferring two genes while controlling the expression doses or expression dose ration thereof.

Description

technical field [0001] The present invention relates to vectors for expressing foreign genes. technical background [0002] Vectors for gene delivery are used in research and gene therapy to express foreign genes in target cells. In such cases, it is sometimes desirable to express both genes in the same target cell. This will allow the target cells into which the therapeutic gene is inserted to selectively proliferate or die by expressing the selective gene in combination with the therapeutic gene. On the other hand, this will allow monitoring the in vivo kinetics of therapeutic transgenic cells by expressing the therapeutic gene in combination with marker genes such as GFP, etc. Furthermore, this would allow the expression of proteins that function by forming complexes between the two types of subunits, such as receptors and transcription factors. [0003] A form in which multiple promoters are inserted and another form in which a promoter is combined with a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N7/01C12N15/67C12N15/867
CPCC12N15/86C12N2830/15C12N2830/00C12N2830/85C12N2830/60C12N2740/15043C12N2840/20A61K48/00C12N15/67C12N15/867
Inventor 中岛俊洋中丸健治长谷川护速水正宪井户荣治
Owner DNAVEC RES
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