Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-02-22
2001-10-02
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025310, C536S025400, C536S025410, C536S025420, C536S023100, C435S006120, C435S259000, C435S267000, C435S268000, C435S269000, C435S270000, C435S810000
Reexamination Certificate
active
06297371
ABSTRACT:
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 To date, part of the cells to be treated, in which the defect gene is expressed, such as blood cells, has been withdrawn from the patients.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 &mgr;g/mg of DNA
Protein
<100 &mgr;g/mg of DNA
Supercoiled DNA
>90%
A
260/280
1.75-1.85
Residual salt
scan from A
220
to A
320
RNA
<1%
Sterility
no colonies after
14 days of
tryptose culture
In addition to the quality of the purified nucleic acid, the scale on which the nucleic acid can be purified is also of crucial importance. Thus, a future technology must enable to purify nucleic acids on a scale of from 1 mg to 100 kg which in turn requires culture volumes of from 1 l to 100 m
3
.
A general problem in the purification of nucleic acids from bacterial cultures is at first the lysis of the microorganisms. In addition to the alkaline lysis described by Birnborn and Dohly (Nucl. Acids Res. 7, pages 1513-1522 (1979)) which is preferred herein, this may also involve the rupture of the bacterial cells by high pressure (French Press), lysis in the presence of detergents, or the application of heat (boiling lysis).
Subsequently, the nucleic acid can be separated more or less effectively from the other components of the bacterial cell, such as proteins or genomic DNA and metabolites, by various methods. The most simple, but also not very efficient, possibility is the separation by the addition of salts, such as LiCl, causing precipitation of the cellular proteins. The nucleic acid can subsequently be precipitated with alcohol. A drawback of this method is that contaminations of RNA, ssDNA and proteins cannot be separated off quantitatively. As an additional purification step, phenol extraction is frequently performed to remove any protein contaminations. The drawback of this method, desingated as “salting out”, is that endotoxin contaminations as well as RNA and ssDNA which may be present cannot be removed. In addition, phenol extraction involves the risk of contaminating the nucleic acid with phenol. Further, phenol treatment of nucleic acids usually results in an increased content of so-called “nicked” nucleic acid, i.e. break of the nucleic acid strand at many sites, which in turn highly affects its stability.
CsCl gradient centrifugation has been an established method for the purification of nucleic acids for nearly 30 years. This makes use of the different sedimentation behaviors of differently sized nucleic acid molecules (RNA, plasmid DNA, genomic DNA) in a CsCl concentration gradient in the presence of intercalating agents, such as ethidium bromide, for the separation of nucleic acids. This type of separation can only be used with large quantities and requires the use of ultracentrifuges. In addition to the high financial expenditure of about DM 60,000.—per ultracentrifuge, another drawback is the considerable expenditure of time of at least 48 h for such a purification. This method achieves a yield of only 5 mg of nucleic acid at most per centrifugal run.
The purification of nucleic acids by chromatographic methods is also known per se. There are generally two types of distinct methods.
Purification by anion exchange chromatography is described in EP 0 268 946 B1. The bacterial cells are preferably lysed by alkaline lysis. The cellular proteins and genomic DNA are separated by means of detergents and subsequent centrifugation. The supernatant thus obtained which contains the plasmid DNA is called the “cleared lysate”. The cleared lysate is further purified over an anion exchange column (QIAGEN®), wherein RNA and ssDNA are quantitatively separated off. Removal of endotoxin does not take place.
Gillespie and Vogelstein, Proc. Natl, Acad. Sci., USA, 76, p. 615-619, state that nucleic acids may be further purified by binding to silica gel or diatomaceous earth in the presence of chaotropic salts, such as GuHCl, NaCl etc. In contrast to anion exchange chromatography, binding of the DNA is here performed in the presence of high salt concentrations whereas elution is performed at low salt concentrations. The mechanism is not understood in all details, but it is considered that the nucleic acid is precipitated by dehydration on the surface of the silica gel particles. Since this involves binding and elution according to an “all-or-none” principle, a quantitative se
Colpan Metin
Moritz Peter
Schorr Joachim
Jacobson & Holman PLLC
Qiagen GmbH
Wilson James O.
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