Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-09
2004-07-13
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S235100, C435S325000, C435S455000, C435S463000, C800S022000
Reexamination Certificate
active
06762038
ABSTRACT:
BACKGROUND OF THE INVENTION
Protein therapeutic agents are among the most important human and animal health products to have emerged from the biotechnology revolution of the 1980's and 1990's. These protein therapeutic agents include interferons, growth factors such as colony stimulating factor and erythropoietin, and monoclonal antibodies.
Many protein therapeutics are produced by a process which involves introducing a nucleic acid which encodes the desired exogenous protein into a living cell, expressing the exogenous protein in the cell, and then recovering the protein, referred to hereinafter as the “recombinant protein” from the system. Although a number of different cell types, including bacteria, yeast, insect cells, and plant cells can be used in this process, there are several advantages to using mammalian cells.
The major advantage to using mammalian cells to produce recombinant proteins is that processing of the proteins is normal. In other words, the proteins which are expressed in mammalian cells typically are glycosylated, phosphorylated and have the signal peptides removed therefrom. As a result, the recombinant protein is more similar to the naturally-occurring protein and, thus, more likely to be biologically active and non-antigenic. In addition, the toxic products that are typically produced in bacteria, yeast, insect cells and plant cells are avoided. Moreover, purification of the recombinant protein is often easier since the processed protein is secreted into the culture medium by mammalian cells. Accordingly, agents such as interferon beta, which is used in treatment of multiple sclerosis, currently are produced in Chinese hamster ovary (CHO)cells.
Unfortunately, high levels of the exogenous protein are not normally produced when mammalian cells are used in the recombinant process. Accordingly, it is desirable to have new methods for enhancing the expression of exogenous proteins in mammalian cells. Immortalized mutant mammalian cell lines which are capable of producing greater amounts of recombinant, exogenous proteins than their corresponding non-mutant mammalian cell lines are also desirable.
SUMMARY OF THE INVENTION
The present invention provides immortalized mutant cell lines that are capable of producing enhanced levels of exogenous, recombinant proteins. The mutant cell lines comprise mammalian somatic cells having a homozygous disruption in the gene which encodes the endoribonuclease known as RNase L and a homygous disruption in the gene which encodes the double-stranded RNA dependent kinase known as PKR. As used herein a “disruption” is a deletion of all or a portion of the RNase L gene or the PKR gene, an addition of one or more nucleotides to the RNase L gene or the PKR gene, a substitution of one or more of the nucleotides in the RNase L gene or the PKR gene, or any combination thereof. Preferably, the disruptions are in a coding exon of the RNase L and PKR genes. As a result of the homozygous disruptions, the mutant cell line, referred to hereinafter as the “double knock-out” or “DKO” cell line lacks biologically active forms of both the RNase L enzyme and the PKR enzyme.
The present invention also provides methods for producing enhanced levels of recombinant proteins, or polypeptides, in mammalian cell systems. In one aspect the method employs cells of the DKO cell line and comprises transfecting the DKO cells with a nucleic acid, or polynucleotide, encoding a desired, exogenous protein, or polypeptide; expressing the exogenous protein in the cell; and isolating the exogenous protein from the system, i.e., from the transfected cells, the culture medium of the transfected cells, or both. Preferably, the DKO cells are also transfected with, i.e., co-transfected with, a nucleic acid encoding the PKR inhibitor, adenovirus VAI RNA.
In another aspect the method employs mutant cells having a homozyous disruption in the RNase L gene alone, referred to hereinafter as “RNase L null” cells. RNase L null cells lack functional RNase L enzyme. The RNase L null cells are transfected with a nucleic acid encoding a desired, exogenous protein. Then the exogenous protein is expressed in the cell and isolated from the system. Preferably, the RNase L cells are co-transfected with a nucleic acid encoding adenovirus VAI RNA, or a nucleic acid encoding a dominant negative PKR, or a combination thereof. The subsequent steps involve expressing the nucleic acids that have been transfected into the RNase L null cells and isolating or purifying the exogenous protein from the system.
In another aspect the methods employ mutant cells having a homozygous disruption in the PKR gene, hereinafter referred to as “PKR null” cells. PKR null cells lack a functional PKR enzyme. In one embodiment, the method comprises co-transfecting the PKR null cells with a nucleic acid encoding adenovirus VAI RNA and with a nucleic acid encoding a desired, exogenous protein, expressing the exogenous protein in the cell, and isolating the exogenous protein from the system. In another embodiment, the method comprises co-transfecting the PKR null cells with a nucleic acid encoding a dominant negative RNase L and with a nucleic acid encoding a desired, exogenous protein; expressing the dominant negative RNase L and the desired, exogenous protein in the cell; and isolating the exogenous protein from the system. Preferably, the PKR null cells are transfected with a nucleic acid encoding adenovirus VAI RNA and a nucleic acid encoding a dominant negative RNase L, as well as with the nucleic acid encoding the exogenous protein.
In another aspect, the method comprises cotransfecting mammalian cells with a nucleic acid encoding the desired exogenous protein and a nucleic acid encoding a dominant negative RNase L or a dominant negative PKR. Preferably, the mammalian cells are cotransfected with both a nucleic acid encoding a dominant negative PKR and a nucleic acid encoding a dominant negative RNase L. More preferably, the mammalian cells are also transfected with a nucleic acid encoding adenovirus VAI RNA. The subsequent steps involve expressing the nucleic acids that have been transfected into the RNase L null cells and isolating the exogenous protein from the system.
The present invention also relates to methods of making the double knock-out cell line.
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Der Sandy
Silverman Robert H.
Terenzi Fulvia
Williams Bryan R. G.
Zhou Aimin
Calfee Halter & Griswold LLP
Chen Shin-Lin
Priebe Scott D.
The Cleveland Clinic Foundation
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