Genomic sequences for protein production and delivery

Details Genomic sequences for protein production and delivery Genomic sequences for protein production and delivery

1999-05-05

2001-06-05

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069600, C435S463000, C435S325000, C435S320100

Reexamination Certificate

active

06242218

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to genomic DNA.
BACKGROUND OF THE INVENTION
Current approaches to treating disease with therapeutic proteins include both administration of proteins produced in vitro and gene therapy. In vitro production of a protein generally involves the introduction of exogenous DNA coding for the protein of interest into appropriate host cells in culture. Gene therapy methods, on the other hand, involve administering to a patient genetically engineered cells, plasmids, viruses that contain a sequence encoding the therapeutic protein of interest.
Certain therapeutic proteins may also be produced by altering the expression of their endogenous genes in a desired manner with gene targeting techniques. See, e.g., U.S. Pat. Nos. 5,641,670, 5,733,761, and 5,272,071, WO 91/06666, WO 91/06667, and WO 90/11354, all of which are incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The present invention is based upon the identification and sequencing of genomic DNA 5′ to the coding sequence of the human granulocyte colony-stimulating factor (“G-CSF”) gene. This DNA can be used, for example, in a DNA construct that alters (e.g., increases) expression of an endogenous G-CSF gene in a mammalian cell upon integration into the genome of the cell via homologous recombination. “Endogenous G-CSF gene” refers to a genomic (i.e., chromosomal) copy of a gene that encodes G-CSF. The construct contains a targeting sequence including or derived from the newly disclosed 5′ noncoding sequence, and a transcriptional regulatory sequence. The transcriptional regulatory sequence preferably differs in sequence from the transcriptional regulatory sequence of the endogenous G-CSF gene. The targeting sequence directs the integration of the regulatory sequence into a region within or upstream of the G-CSF-coding sequences of the target gene such that the regulatory sequence becomes operatively linked to the endogenous coding sequence. By “operatively linked” is meant that the regulatory sequence can direct expression of the endogenous G-CSF-coding sequence. The construct may additionally contain a selectable marker gene to facilitate selection of cells that have stably integrated the construct, and/or another coding sequence operatively linked to a promoter.
In one embodiment, the DNA construct contains: (a) a targeting sequence, (b) a regulatory sequence, (c) an exon, and (d) a splice-donor site. The targeting sequence directs the integration of itself and elements (b)-(d) into a region within or upstream of the G-CSF-coding sequences of the target gene. Once integrated, element (b) can direct transcription of elements (c) and (d) and all downstream coding sequences of the endogenous gene. In the construct, the exon is generally 3′ of the regulatory sequence, and the splice-donor site is at the 3′ end of the exon.
In another embodiment, the DNA construct comprises: (a) a targeting sequence, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of itself and elements (b)-(f) such that elements (b)-(f) are within or upstream of the endogenous gene. The regulatory sequences then directs production of a transcript that includes not only elements (c)-(f), but also endogenous G-CSF coding sequences. Preferably, the construct-derived intron and splice-acceptor site are situated in the construct downstream from the splice-donor site.
The targeting sequence is homologous to a pre-selected target site in the genome with which homologous recombination is to occur. It contains at least 20 (e.g., at least 50 or 100) contiguous nucleotides from SEQ ID NO:5, which represents nucleotides −6578 to −364 relative to the translation start site of the human G-CSF gene. By “homologous” is meant that the targeting sequence is identical or sufficiently similar to its genomic target site so that the targeting sequence and target site can undergo homologous recombination. A small percentage of basepair mismatches is acceptable, as long as homologous recombination can occur at a useful frequency. To facilitate homologous recombination, the targeting sequence is preferably at least about 20 (e.g., 50, 100, 250, 400, or 1,000) base pairs (“bp”) long. The targeting sequence can also include genomic sequences from outside the region covered by SEQ ID NO:5, so long as it includes at least 20 nucleotides from within this region. For example, additional targeting sequence could be derived from the sequence lying between SEQ ID NO:5 and the endogenous transcription initiation sequence of the G-CSF gene.
Due to polymorphism that may exist at the G-CSF genetic locus, minor variations in the nucleotide composition of any given genomic target site may occur in any given mammalian species. Targeting sequences that correspond to such polymorphic variants of SEQ ID NO:5 (particularly human polymorphic variants) are within the scope of this invention.
Upon homologous recombination, the regulatory sequence of the construct is integrated into a pre-selected region upstream of the coding sequence of a G-CSF gene in a chromosome of a cell. The resulting new transcription unit containing the construct-derived regulatory sequence alters the expression of the target G-CSF gene. The G-CSF protein so produced may be identical in sequence to the G-CSF protein encoded by the unaltered, endogenous gene, or may contain additional, substituted, or fewer amino acid residues as compared to the wild type G-CSF protein, due to changes introduced as a result of homologous recombination.
Altering gene expression encompasses activating (or causing to be expressed) a gene which is normally silent (i.e, essentially unexpressed) in the cell as obtained, increasing or decreasing the expression level of a gene, and changing the regulation pattern of a gene such that the pattern is different from that in the cell as obtained. “Cell as obtained” refers to the cell prior to homologous recombination.
Also within the scope of the invention is a method of using the present DNA construct to alter expression of an endogenous G-CSF gene in a mammalian cell. This method includes the steps of (i) introducing the DNA construct into the mammalian cell, (ii) maintaining the cell under conditions that permit homologous recombination to occur between the construct and a genomic target site homologous to the targeting sequence, to produce a homologously recombinant cell; and (iii) maintaining the homologously recombinant cell under conditions that permit expression of the G-CSF coding sequence under the control of the construct-derived regulatory sequence. At least a part of the genomic target site is 5′ to the coding sequence of an endogenous G-CSF gene. That is, the genomic target site can contain coding sequence as well as 5′ non-coding sequence.
The invention also features transfected or infected cells in which the construct has undergone homologous recombination with genomic DNA upstream of the endogenous ATG initiation codon in one or both alleles of the endogenous G-CSF gene. Such transfected or infected cells, also called homologously recombinant cells, have an altered G-CSF expression pattern. These cells are particularly useful for in vitro G-CSF production and for delivering G-CSF via gene therapy. Methods of making and using such cells are also embraced by the invention. The cells can be of vertebrate origin such as mammalian (e.g., human, non-human primate, cow, pig, horse, goat, sheep, cat, dog, rabbit, mouse, guinea pig, hamster, or rat) origin.
The invention further relates to a method of producing a mammalian G-CSF protein in vitro or in vivo by introducing the above-described construct into the genome of a host cell via homologous recombination. The homologously recombinant cell is then maintained under conditions that allow transcription, translation, and optionally, secretion of the G-CSF protein.
The invention also features an isolated nucleic acid comprising a seque

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