Process for the separation and purification of nucleic acids fro

Details Process for the separation and purification of nucleic acids fro Process for the separation and purification of nucleic acids fro

1996-10-18

1999-11-23

Wilson, James O.

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

536 253, 536 2531, 536 2541, 536 2542, 435 6, 435259, 435267, 435270, 435810, C07H 2100, C12N 108

Patent

active

059903015

DESCRIPTION:

BRIEF SUMMARY
The present invention pertains to a process for the isolation and purification of nucleic acids and/or oligonucleotides for use in gene therapy wherein said nucleic acids and/or oligonucleotides are purified from an essentially biological source, the use of anion exchange materials for the separation, purification and isolation of nucleic acids for the preparation of an agent containing nucleic acids for gene therapy, and a kit containing components for performing the process according to the invention.
A new form of therapy for genetically caused diseases, such as cystic fibrosis or muscular dystrophy, is based on the discovery that such diseases are caused by particular genetic defects. A therapy for the genetic defect appears to be possible if the healthy gene is supplied to the afflicted organism in a sufficient amount. Gene therapy not only enables the treatment of genetically caused diseases, but is also suitable for the treatment of tumors, and is suited as a new form of inoculation against infectious diseases, such as hepatitis, influenza, and HIV, to give but a few examples (TIBTECH, Special Issue: Gene Therapy Therapeutic Strategy and Commercial Prospects, May 1993, Vol. 11, No. 5 (112)).
A central problem of gene therapy is to administer the therapeutic DNA in such a manner that it will reach the scene of action. To date, part of the cells to be treated, in which the defect gene is expressed, such as blood cells, has been withdrawn from the patients. These cells have been cultured in culture dishs (in vitro). In order to introduce the therapeutically active foreign DNA into the cells, gene segments of a retrovirus, e.g., have been used which were linked to the DNA to be introduced. The genetically altered cells have been retransferred into the organism (Anderson, W. F. (1992), Human Gene Therapy, Science 256: 808-813).
Currently, a number of clinical studies are already being performed with this so-called ex vivo approach. This has lately involved the use of plasmid DNA, oligonucleotides, mRNA, genomic DNA, YACs (yeast artificial chromosomes), in addition to the retroviruses mentioned above, for the transfection of cell cultures. However, the ex vivo method involves a high expenditure of work and is not suited for the treatment of all diseases. There may be mentioned, for example, muscular dystrophy or cystic fibrosis. Thus, it is desirable to provide simpler procedures to administer therapeutically useful DNA to an organism. It has been found in this context that it is possible to administer plasmid DNA directly into the tissue of an organ. Part of the DNA will be transported to the nucleus. The genetic information administered via the DNA is translated there into the therapeutically active protein. The treatment within the organisms is a direct one and is called in vivo treatment.
For in vivo treatment, the DNA or RNA may also be mixed with liposomes or other substances, resulting in a better intake of the nucleic acids into the cell. However, the nucleic acid may also be directly injected into the organ to be treated, for example, a muscle or a tumor (Plautz, G. E. et al., 1993, PNAS, Vol. 90, 4645-4649). The advantage is that the DNA entering the organism does not cause any immunological reactions in the organism if it is free of accompanying immunogenic contaminations. Therefore, in vivo gene therapy makes high demands on the quality of the nucleic acids to be administered. The DNA must be free of toxic substances which might result in pathogenic effects in the organism to be treated. Clinical phase I studies on humans using this technology have resulted in rather detailed and strict requirements for the nucleic acids used therein. According to the requirements of the FDA in the U.S.A., the nucleic acids employed for therapeutical uses have to pass the following quality controls:


______________________________________ Examination of the nucleic acid for: requirement/limit ______________________________________ Endotoxins <300 I.U./mg of DNA E. coli genomic DNA <50 .mu.g/mg o

REFERENCES:
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patent: 5057426 (1991-10-01), Henco et al.
patent: 5652141 (1997-07-01), Henco et al.
patent: 5747663 (1998-05-01), Colpan et al.
Patent Abstract of Japan, Derwent Publications Ltd., JPA58013519 (Seikagaku Kogyo KK), Jan. 26, 1983.

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