Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Treating animal or plant material or micro-organism
Reexamination Certificate
1999-04-14
2004-08-24
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Treating animal or plant material or micro-organism
C435S019000, C435S199000, C435S041000, C435S069100, C435S072000, C435S091100, C530S350000, C536S023200
Reexamination Certificate
active
06780632
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the production of RNA free cellular components, and a method of removing RNA from preparations of cellular components.
BACKGROUND OF THE INVENTION
The ribonuclease enzyme family (hereafter referred to as RNases) has been extensively studied. Numerous RNases have been characterized and the genes encoding some of these proteins have been cloned.
RNases hydrolyze one or more of the phosphodiester bonds in single stranded and double stranded RNA as well as RNA in RNA:DNA hybrids. RNases differ in their specificity for either a particular form of the RNA substrate (for example single stranded, double stranded or in a DNA:RNA hybrid) or their specific point of RNA cleavage.
One biological activity of RNase enzymes is the processing of mature molecules of RNA from precursor forms (Genes, Benjamin Lewin ed., John Wiley & Sons, 2nd edition, p.395, 1985). Certain RNases can affect the growth and differentiation of mammalian cells by virtue of an intrinsic anti-tumor activity (references within Ribo et al., Prot. Express. and Purif. 7: 253-261, 1996).
RNA is a major contaminant of preparations from cell lysates. For example, plasmid DNA preparations contain RNA that is difficult to remove by anion exchange or size exclusion chromatography because it is similar in size (with respect to exclusion by SEC matrices) and charge to DNA. RNase A hydrolyzes RNA after C and U residues by cleaving between the 3′-phosphate group of a pyrimidine ribonucleotide and the 5′-hydroxyl of the adjacent nucleotide. This enzyme is used to degrade RNA to low molecular weight species that no longer copurify with plasmid or genomic DNA. RNase I can also be used to remove RNA from preparations of plasmid or genomic DNA. RNase A is also commonly used for the enzymatic digestion of host derived RNA during the production of recombinant protein from
E. coli.
A prior art method of removing RNA from a sample is to add a large quantity of an exogenously produced RNase. For example, to remove RNA from plasmid DNA bovine RNase A is added to a final concentration of 100 &mgr;g/ml (QIAGEN Plasmid Handbook February 1995, QIAGEN Ltd. Unit 1 Tillingbourne Court, Dorking Business Park, Dorking, Surrey RH4 1HJ, UK). Bovine RNase A is commonly used at a final concentration of approximately 10-100 &mgr;g/ml for the enzymatic digestion of host derived RNA during the production of recombinant protein from
E. coli
. A chief disadvantage of prior art methods is that the cellular component that is treated according to these methods often contains residual RNase of animal origin.
There are limitations to using exogenously produced RNases. First, it is difficult to purify large amounts of RNase from the tissue of origin. This is presumably because high concentrations of active RNase will degrade host cell RNA and impair normal cell functions to a level that can be toxic to a cell. Therefore, it is difficult to produce large quantities of active RNase by expression in cells because high concentrations of active RNase will be toxic to a cell. It is also difficult to produce large quantities of active RNase by expression in cells because RNase can be sensitive to proteases of the host in which it is being synthesized, and because RNases that are over-produced and accumulate as in e.g.
E. coli
inclusion bodies cannot always be correctly refolded. The most significant limitation to using exogenously produced RNase is that, if the exogenously added RNase is of animal origin, following RNase treatment the presence of residual enzyme can contaminate a DNA preparation, thereby rendering it unacceptable for certain applications, including gene therapy. Third, it is expensive to produce RNase in large quantities.
There is a need in the art for plasmid DNA and protein preparations that are substantially free from contaminating RNA.
There is a need in the art for methods of producing RNA-free cellular components that are suitable for administration to human subjects.
There is a need in the art for a method of removing RNA from cellular components that does not rely on incubating a cellular lysate or purified component with added exogenously produced RNase.
SUMMARY OF THE INVENTION
The invention features a method of preparing a substantially RNA-free cellular component comprising culturing cells producing the cellular component in a medium and lysing said cells to produce a cell lysate, wherein said cell lysate contains said cellular component and sufficient RNase activity to degrade substantially all of the RNA molecules present in said cell lysate.
In a preferred embodiment, the RNase is produced by cells producing the cellular component.
Alternatively, the RNase is produced by cells in the medium other than those cells producing the cellular component.
The invention also features a method of preparing a substantially RNA-free cellular component, comprising culturing and lysing cells producing the cellular component and cells producing an RNase in an amount sufficient to degrade substantially all of the RNA present in the preparation.
Preferably, the cells producing the cellular component also produce the RNase and the culture and lysate contain cells producing the cellular component and an RNase in an amount sufficient to degrade substantially all of the RNA present in the preparation. Preferably, the cellular component and the RNase are produced by the same cell.
In preferred embodiments of both of the above-described inventive methods, the cellular component is one of a DNA, a protein, and a carbohydrate. Preferably, the cellular component is one of a recombinant DNA, a recombinant protein and a recombinant carbohydrate. Preferably, the RNase is encoded on a plasmid and the cellular component is encoded on the same plasmid, another plasmid or on the cell's chromosome.
In preferred embodiments of both of the above-described inventive methods the gene encoding said RNase is integrated into the genome of the cell producing the RNase.
In some methods of the invention, it is preferred that the RNase is non-specific. Such a non-specific RNase may be RNase A, RNase M of RNase I.
In preferred embodiments of both of the above-described inventive methods, a cell producing an RNase produces the RNase in a regulated manner.
Preferably, the RNase produced by the host cell is overproduced, either by inducible production, or by constitutive production. The RNase overproduced by the host cell also may be secreted out of the host cell cytoplasm, for example, secreted into the host cell periplasm or secreted out of the host cell into the medium.
In some methods, it is preferred that the RNase is a non-specific RNase.
The invention also features a composition comprising a host cell that produces a recombinant DNA, a recombinant protein, or a recombinant carbohydrate and also produces an RNase in a regulated manner.
RNase produced in said regulated manner is overproduced, or inducibly overproduced, or constitutively overproduced. The RNase produced by the host cell also may be secreted out of the host cell cytoplasm, for example, secreted into the host cell periplasm or secreted out of the host cell into the medium.
In some methods, it is preferred that the RNase is a non-specific RNase.
The invention also features a composition comprising a host cell that produces a recombinant DNA, a recombinant protein, or a recombinant carbohydrate and a host cell that produces an RNase in a regulated manner.
RNase produced in said regulated manner is overproduced, or inducibly overproduced, or constitutively overproduced. The RNase produced by the host cell also may be secreted out of the host cell cytoplasm, for example, secreted into the host cell periplasm or secreted out of the host cell into the medium.
The invention also features a pharmaceutical composition comprising a cellular component that is substantially RNA-free, in a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition comprising a cellular component that is substantially RNA-free obtainable by
Hanak Julian Alexis John
Williams Steven Geraint
Cobra Biologics Limited
Cozen O'Connor P.C.
Prouty Rebecca E.
Ramirez Delia
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