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
2000-12-11
2003-06-10
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120
Reexamination Certificate
active
06576443
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for modifying the genomes of eukaryotic cells to alter the expression of selected endogenous genes. The invention also relates to polypeptides and recombinant eukaryotic host cells produced by the practice of the disclosed methods. The invention further relates to vector systems useful for modifying the genomes of eukaryotic cells to alter the expression of selected endogenous genes.
2. Related Art
A considerable number of diseases are based on impaired gene regulation leading to the loss of appropriate responses to certain physiological conditions due to lack of production of appropriate factors (e.g., hormones, blood clotting factors). A number of factors have been isolated in the past from organs and bodily fluids, such as urine, for use in the treatment of such diseases. Several disadvantages are implicit to such isolation regimens. First, relatively large amounts of raw material are required to obtain relatively small amounts of purified substance. Secondly, when the factors are derived from non-human organisms, they often elicited undesired side-reactions upon administration to humans.
Progress in gene technology has allowed for the cloning of genes and the in vitro production of encoded proteins. These production processes are generally performed by cloning the gene of interest into an appropriate expression vector and introducing the latter into suitable cells.
An alternative approach to producing gene products is to directly increase expression of the endogenous gene in an appropriate cell line. This approach obviates the necessity of isolating the endogenous gene from the cell in which it resides.
Gene expression can be modified at several levels, e.g., by manipulating the efficiencies of transcription, translation, protein folding or secretion. We present methods for increasing expression of endogenous genes by keeping transcription efficiency constant but increasing the amount of mRNA within the cell using RNA replication.
SUMMARY OF THE INVENTION
The present invention provides both methods and nucleic acid constructs for altering the expression characteristics of endogenous target genes in eukaryotic cells. Further provided are recombinant eukaryotic host cells which are produced by the methods of the invention and gene expression products produced using the recombinant eukaryotic host cells.
In particular, the invention provides methods for modifying the expression characteristics of endogenous target genes within the genomes of eukaryotic cells comprising inserting into these genomes exogenous nucleic acid which encodes genetic elements required for RNA replication and amplifying RNA corresponding to the coding region of the endogenous target genes.
In one general aspect, the invention provides methods for modifying the expression characteristics of endogenous target genes within the genome of eukaryotic cells comprising inserting exogenous polynucleotides in the 5′ and 3′ regions flanking the endogenous target genes to produce recombinant eukaryotic host cells, and culturing the recombinant eukaryotic host cell under conditions which allow for transcription and replication of RNA corresponding to the endogenous target genes, wherein the exogenous polynucleotides encode genetic elements required for RNA replication.
In another general aspect, the invention provides methods for producing polypeptides encoded by endogenous target genes of eukaryotic cells comprising inserting exogenous polynucleotides in the 5′ and 3′ regions flanking the endogenous target genes to produce recombinant eukaryotic host cells, and culturing the recombinant eukaryotic host cells under conditions which allow for transcription, replication, and translation of RNA corresponding to the endogenous target genes, wherein the exogenous polynucleotides encode genetic elements required for RNA replication which alter the expression characteristics of the endogenous target genes.
In a related aspect, the exogenous polynucleotides used in the methods of the invention contain one or more positive or negative selection markers (e.g., neomycin phosphotransferase, metallothionein I, metallothionein II, dihydrofolate reductase, hygromycin B phosphotransferase, puromycin-N-acetyl-transferase, xanthine/guanine phosphoribosyl transferase, histidinol dehydrogenase, Herpes simplex thymidine kinase, cytosine deaminase, Diptheria toxin, and hypoxanthine phosphoribosyl transferase).
Further, these selection markers may be operably linked to a viral subgenomic promoter or an RNA polymerase II promoter.
In addition, when the selection marker is a positive selection marker, this marker may be co-transcribed with the coding region of the endogenous target gene as part of the same RNA molecule and translated from an internal ribosome entry site.
Further, when negative selection markers are used, these markers will generally be positioned in the exogenous polynucleotides such that they are excised when integration occurs by homologous recombination.
In another related aspect, the exogenous polynucleotides which encode genetic elements required for RNA replication are derived from an Alphavirus.
In yet another related aspect, the methods of the invention result in the production of recombinant eukaryotic host cells wherein the endogenous target genes with modified expression characteristics are operably linked to an Alphavirus subgenomic promoter.
In further related aspects the recombinant eukaryotic host cells generated by the methods of the invention are animal cells, vertebrate cells, mammalian cells, and/or human cells. Further, when the recombinant eukaryotic host cells are human cells, these cells may be derived from organs such as the kidney, liver, or testes. In addition, when the recombinant eukaryotic host cells are human liver cells, these cells may be Hep G2 cells, Hep 3B cells, or a cell type which is a derivative thereof. In addition, when the recombinant eukaryotic host cells are human kidney cells, these cells may be 293 cells, or a cell type which is a derivative thereof.
The invention further relates to methods for modifying the expression characteristics of selected endogenous genes, referred to herein as endogenous target genes, which encode ribozymes and polypeptides. When the expression of an endogenous target gene is modified by the methods of the invention, this gene may encode a human polypeptide such as erythropoietin, antithrombin III. &agr;-galactosidase, granulocyte-macrophage colony-stimulating factor. megakaryocyte-growth factor (M-GF), blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, &agr;-interferon, &bgr;-interferon, &ggr;-interferon, interleukin-2, tissue plasminogen activator, thrombopoietin, &agr; I-antitrypsin, LDL-receptor, insulin, or growth hormone.
The invention also relates to recombinant eukaryotic host cells produced by the methods of the invention.
In another general aspect, the invention provides DNA vector systems for modifying the expression characteristics of selected endogenous genes within the genomes of eukaryotic cells. These vector systems comprise 5′ targeting constructs and 3′ targeting constructs which contain genetic elements required for RNA replication.
In a related aspect, the 5′ and 3′ targeting constructs of the vector systems of the invention contain genetic elements of an Alphavirus. Further, these genetic elements may be derived from Semliki Forest Virus, Sindbis virus, Venezuelan equine encephalomyelitis virus, or Ross River Virus.
In another related aspect, the 5′ and 3′ targeting constructs of the vector systems of the invention may contain one or more positive or negative selection markers (e.g., neomycin phosphotransferase, metallothionein I, metallothionein II, dihydrofolate reductase, hygromycin B phosphotransferase. puromycin-N-acetyl-transferase, xanthine/guanine phosphoribosyl transferase, histidinol dehydrogenase, Herpes simplex thymidine kinase, cytosine
Hennecke Frank
Renner Wolfgang A.
Cytos Biotechnology AG
Horlick Kenneth R.
Sterne Kessler Goldstein & Fox PLLC
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