Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-08-04
2004-06-15
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S471000, C435S477000, C536S025400
Reexamination Certificate
active
06750333
ABSTRACT:
The invention concerns the preparation of purified nucleic acid and its use especially in gene therapy.
Replicatable nucleic acid is usually produced by amplifying replicatable plasmid DNA in gram-negative bacteria such as e.g.
E. coli
. After lysis of the biomass (usually alkaline lysis with lysozyme or ultrasound), it is centrifuged and the supernatant is shaken out with phenol. Subsequently an ultracentrifugation on a caesium chloride gradient is carried out (Birnboim & Doly, Nucleic Acid Res. 7 (1979) 1513-1523, Garger et al., Biochem. Biophys. Res. Comm. 117 (1983) 835-842). However, such preparations contain endotoxins, phenol, caesium chloride and/or ethidium bromide as a dye.
A further process is described in the QIAGEN® Plasmid Handbook (Qiagen Inc., Chatsworth, USA) and EP-B 0 268 946. According to this process the cell lysate obtained after a conventional lysis is chromatographed on QIAGEN®-TIP, which contains QIAGEN® resin (a support material based on silica). The disadvantage of this process is that DNA binding proteins are not completely detached from the DNA and therefore the purified plasmid fraction contains proteins and in particular endotoxins (from the membrane of the gram-negative host cells) in considerable amounts. In another process after alkaline lysis of the
E. coli
biomass the centrifugation supernatant is chromatographed according to Birnboim & Doly under high salt conditions over anion exchange columns (e.g. Mono-Q, Source-Q from Pharmacia, Macroprep-Q from BioRad, Poros-Q from Perseptive Biosystems or HyperD-Q from Biosepra, cf. Chandra et.al., Analyt. Biochem. 203 (1992) 169-172; Dion et al., J. Chrom. 535 (1990) 127-147). Also in this case the purified plasmid fraction contains proteins and in particular endotoxins in considerable amounts.
In another process after alkaline lysis and subsequent phenol/chloroform extraction it is possible to chromatograph by gel filtration (McClung & Gonzales, Analyt. Biochem. 177 (1989) 378-382; Raymond et al., Analyt. Biochem. 173 (1988) 125-133). Even after this purification the plasmid preparation contains impurities and in particular phenol.
A process for the isolation and purification of nucleic acids for use in gene therapy is described in WO 95/21177 in which the purification is essentially carried out by centrifugation, filtration, affinity chromatography or chromatography on an inorganic chromatographic material with subsequent chromatography on an ion exchanger. An additional removal of endotoxins can then be achieved according to WO 95/21177 when the nucleic acid is treated with an endotoxin removal buffer which contains 10% Triton®X100 and 40 mmol/1 MOPS buffer (3-morpholino-1propanesulfonate buffer). A disadvantage of this process is that the nucleic acid purified in this manner contains impurities of Triton® and MOPS buffer. Although endotoxins can be removed by this process to a content of ca. 100 EU/mg DNA (Qiagen News 1/96, 3-5), it is not possible to remove endotoxins to a greater extent by this process.
However, for a therapeutic application such as for example for gene therapy a nucleic acid preparation is required which is as free as possible of all impurities (in particular substantially free of endotoxins). Above all the endotoxin content of plasmid preparations has been hitherto an unsolved problem as described for example by Cotten et al., Gene Therapy 1 (1994) 239-246. A reduced endotoxin content (ca. 100 EU/mg DNA) can only be achieved by the state of the art such as for example according to WO 95/21177 if the nucleic acids are treated with non-ionic detergents such as e.g. Triton (endotoxin removal buffer from WO 95/21177). However, Triton® has a biological action such as e.g. lung changes or reduction of blood pressure (Fiedler, “Lexikon der Hilfstoffe für Pharmazie und Kosmetik und angrenzende Gebiete (Band 9, 3rd edition, 1989, Editio Cantor, Del.)). The MOPS buffer which is additionally required also contains a substance that is problematic with regard to a therapeutic application.
The invention provides a nucleic acid preparation, preferably a plasmid DNA, of high purity in which endotoxins are substantially removed and preferably without ethidium bromide, phenol, caesium chloride, polymyxin or non-ionic detergents and also provides a simple and effective process for purifying such nucleic acids in particular for removing endotoxins.
The invention concerns a nucleic acid that can be replicated in gram-negative bacteria, preferably a plasmid DNA with a content of less than 1% protein, preferably less than 0.1% protein and a content of less than 1 EU/mg DNA, preferably 0.01-0.1 EU/mg DNA of endotoxins. This plasmid DNA is preferably free of ethidium bromide, phenol and caesium chloride, free of detergents based on octylphenolpoly(ethylene glycol ether)
n
such as Triton® detergents and also free of MOPS buffer substance and RNAse.
Amplification is understood as an increase in the copy number of a nucleic acid (in particular DNA and plasmid DNA) based on the replication of a vector. In this process numerous copies are produced from a template. A vector is replicated which represents the nucleic acid or which contains the cloned nucleic acid.
A plasmid DNA is understood as an extrachromosomal DNA duplex molecule. The size of a DNA plasmid is usually 1 to more than 200 kb and one to several hundred copies are present in host cells. Plasmid DNA is usually amplified in gram-negative bacteria such as e.g.
E.coli
and subsequently isolated. Plasmids are often used to construct cloning vectors, for the transfection of prokaryotic and eukaryotic cells. A therapeutic use is of especial importance inconnection with in vivo and ex vivo gene therapy. Plasmid DNA that is used therapeutically preferably has a length of 5 to 20 kb, particularly preferably 5-10 kb and is double-stranded. The plasmid DNA can be linearized or circularly closed. Preferably DNA is used that is essentially circularly closed.
Consequently the invention additionally concerns a pharmaceutical composition containing a nucleic acid according to the invention, preferably plasmid DNA, in a therapeutically effective amount and optionally additional pharmaceutical auxiliary substances, fillers or additives.
Endotoxins are lipopolysaccharides from gram-negative bacteria. Endotoxins can have a pyrogenic effect in mammals and induce an endotoxin shock. The main toxic component of endotoxins is lipid A, the polysaccharide moiety mediating the water solubility and the lipid moiety having the toxic effect. The biological effect of endotoxins in mammals are in particular a hypersensitization as well as other reactions which are accompanied by fever.
Plasmid DNA is amplified by standard methods in
E. coli
i.e. a gram-negative bacterium. After fermentation the biomass obtained in this process is lysed and the cells are lysed. In this process the endotoxins are released from the cell membrane. This means that after amplification of nucleic acids, in particular of plasmid DNA, in gram-negative bacteria and in particular in
E. coli
it is necessary to remove endotoxins if it is intended to use this plasmid DNA therapeutically.
Depending on the application doses of 50 &mgr;g to 10 mg and more are used or planned for a therapeutic application of replicatable nucleic acids, in particular of plasmid DNA. The dose amount depends on the disease and type of administration. In an aerosol, e.g. for the treatment of cystic fibrosis, doses of 400 &mgr;g and more are used. This applies likewise to plasmid DNA encapsulated in a lipid complex (e.g. in liposomes). In order to provide such amounts of replicatable nucleic acid that can be used therapeutically, it is necessary to produce the replicatable nucleic acid on a large scale. For this fermentation preparations are expedient with 1-5 kg biomass from which 1-5 g nucleic acid can be isolated.
The invention also concerns a process for the production of a plasmid DNA with a content of less than 1 EU/mg DNA, preferably 0.01-0.1 EU/mg DNA of endotoxins which is characterized in that plasmid DNA is r
Ketter James
Roche Diagnostics GmbH
Rothwell Figg Ernst & Manbeck P.C.
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