Process for preparing retrovirus vector for gene therapy

Abstract

The present invention provides a process for preparing a retrovirus to be expressed at a high titer by specifically transferring a desired foreign gene into target cells. A pseudotyped retrovirus vector having a high titer can be prepared by transferring a DNA construction wherein a promoter, an loxP sequence, a VSV-G gene and a polyA addition signal are arranged in this order is transferred into cells carrying the retrovirus gag and pol gene expression systems, and then transferring a retrovirus vector containing the desired foreign gene thereinto, followed by the treatment with a recombinase.

Description

[0001] 1. Technical Field[0002] The present invention relates to a process for preparing a retrovirus vector having a high titer and employed in gene therapy.[0003] 2. Prior Art[0004] Owing to the remarkable progress in genetic engineering in recent years, there have been identified genes causative of a number of genetic diseases and thus the pathological mechanisms of these diseases have been clarified at the molecular level. Under these circumstances, studies on gene therapy have been made to transfer genes seemingly capable of ameliorating diseases into cells and some of these treatments have been already put into practical use. Also, attempts have been made to apply the gene therapy to the treatment of cancer, AIDS, etc. In the gene therapy, there are known several methods for transferring foreign genes. Among all, the most frequently employed method at the present stage is the one with the use of retrovirus vectors (Miller, A. D., Blood, 76, 271-278, 1990). Use of these vectors...

AI Technical Summary

Benefits of technology

[0013] Under these circumstances, an object of the present invention is to establish a process whereby a retrovirus vector for specifically transferring foreign genes including those affecting cells into target cells over a broad range and expressing the genes therein can be stably produced at a high titer by strictly regulating, compared with the conventional processes, cytotoxic virus structural proteins; to establish a process for elevating the recovery yield of a retrovirus vector by inhibiting the reinfection of producing cells with a pseudotyped retrovirus vector; and to efficiently screen high-expression cell clones by using a low-efficient drug resistance gene or a short-lived transcript drug resistance gene in the transcription of two genes by a recombinase with the use of the same promoter.

Problems solved by technology

On the other hand, this method suffers from some problems such that no gene can be transferred into cells in a number of cases because of the absence of any receptor for virus envelope proteins in the target cells, that large-sized DNAs cannot be inserted thereby, and that the gene transfer thereby is available exclusively into cells which are capable of dividing.

Although gene therapy with the use of retroviruses has been frequently employed, no sufficient therapeutic effect can be achieved thereby hitherto because of the above problems (Marshall, E., Science, 269, 1050-1055, 1995).

However, it is difficult to prepare vectors with such high qualities as usable for clinical purposes by this process.

In spite of these efforts, no technique has been established so far for a vector having a broad infection range and a high titer in a large scale stably, which is one of serious obstacles to the gene therapy.

However, it is difficult to produce the VSV-G gene product at a high level and at a high reproducibility in packaging cells, since the VSV-G gene product per se has a cytotoxicity.

This is a serious problem in the development of pseudotyped vectors which are expected to be widely applicable.

However, there still remain some problems in these reports such that the regulation of the expression of the VSV-G gene product was not completely regulated by tetracycline and, therefore, the producing cells might be still re-infected with about 10.sup.2 to 10.sup.4 i.u. / ml of the VSV-G pseudotyped virus vector thus produced; and that the stability of the packaging cells over a long period of time was still unreliable, since they were made of the co-transfection of a DNA with the VSV-G expression and another DNA with the drug-resistance gene expression.

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