Recombinant human immunodeficiency virus producing cell lines

Details Recombinant human immunodeficiency virus producing cell lines Recombinant human immunodeficiency virus producing cell lines

1997-09-12

2000-04-11

Stucker, Jeffrey

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Primate cell, per se

4351721, 4351723, 4353201, 424 932, 424 9321, 424 936, 424 937, 935 22, 935 23, 935 32, 935 55, 935 56, 935 57, 935 66, 935 70, 935 71, C12N 1500

Patent

active

060487259

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

This invention relates to recombinant human immunodeficiency virus vectors (hereinafter, HIV vectors), a process for producing said vectors, and productive cells capable of sustaining said vectors stably.


BACKGROUND ART

The recent rapid advances in genetic engineering triggered the development of various techniques in molecular biology. This has been commensurate with remarkable advances in the analysis of genetic information and the unravelling of functions of genes and many attempts are being made to exploit these achievements in practical therapeutic settings. One of the areas that have seen the most remarkable advances is that of gene therapy. Etiological genes of various genetic diseases have been discovered and deciphered on one hand, and procedures for transferring such genes into cells by physical and chemical techniques have been developed on the other; as a result, gene therapy has progressed from the stage of preclinical experimentation to practical clinical applications.
Depending on the type of cells (target cells) for gene transfer, gene therapy is classified as either germline cell gene therapy or somatic cell gene therapy. Another way of classification is into augmentation gene therapy which involves the addition of a new (normal) gene, with an abnormal (etiological) gene left intact, and replacement gene therapy for replacing an abnormal gene by a normal gene. At the present stage, only augmentation gene therapy of somatic cells is being practised in consideration of ethical and technological restraints. More specifically, a method of gene therapy in current practice is one by autotransplantation (ex vivo gene therapy) in which a target cell is taken out of the patient and a gene to be inserted is transferred into the target cell, which is then replaced into the patient's body. A method under review for future possibility is one that involves direct gene administration into the patient (in vivo gene therapy).
One of the major technological challenges for the clinical application of the above-described gene therapy is the development of a method for introducing an exogenous gene into a target cell in an efficient and consistent way. In the early eighties, physical techniques such as microinjection were attempted; however, these methods were not eventually commercialized for several reasons such as low gene transfer efficiency, incapability to achieve consistent transfer and the limitations of the then available technology of large-scale cell cultivation. It was not until a recombinant virus (viral vector) for inserting an exogenous gene efficiently into a target cell was later developed that the clinical application of gene therapy became a possibility.
In the United States of America, about 70 protocols of gene therapy have been approved and in practice no fewer than 200 patients are presently undergoing gene therapy. A mouse leukemia virus (MoMLV, or Moloney's murine leukemia virus) vector is the most commonly used means of gene transfer. MoMLV is a kind of retrovirus and infects a host cell if the envelope on its surface binds specifically to the receptor on the surface of the host cell. Recombinant MoMLVs are capable of gene transfer into different cell species depending on the type of envelope and are classified as, for example, ecotropics which infect only rodents and amphotropics which infect both rodents and human cells.
Preparing a recombinant MoMLV vector first requires the construction of two plasmids, one being a helper plasmid which comprises gag, pol and env genes to be coded in a MoMLV genome and a promoter for driving these genes, and the other being a vector plasmid having the terminal repeated sequence (LTR) of MoMLV inserted at both ends of the gene acting as a drug. In this case, for the purpose of preventing the production of a wild type of virus, a packaging signal which is a signal sequence for packaging the gene into the particle of a virus is preliminarily removed from the helper plasmid. In contrast, the packaging signal is contained in the vect

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