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Novel method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same

Inactive Publication Date: 2009-02-19
IDEC PHARM CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0050]Based on the foregoing, it is apparent that a significant advantage of the invention is that it substantially reduces the number of colonies that need be screened to identify high producer clones, i.e., cell lines containing a desired DNA which secrete the corresponding protein at high levels. On average, clones containing integrated desired DNA may be identified by screening about 5 to 20 colonies (compared to several thousand which must be screened when using standard random integration techniques, or several hundred using the previously described intronic insertion vectors) Additionally, as the site of integration was preselected and comprises a transcriptionally active domain, all exogenous DNA expressed at this site should produce comparable, i.e. high levels of the protein of interest.
[0051]Moreover, the subject invention is further advantageous in that it enables an amplifiable gene to be inserted on integration of the marking vector. Thus, when a desired gene is targeted to this site via. homologous recombination, the subject invention allows for expression of the gene to be further enhanced by gene amplification. In this regard, it has been reported in from the literature that different genomic sites e different capacities for gene amplification (Meinkoth et al, Mol. Cell Biol., 7:1415-1424 (1987)). Therefore, this technique is further advantageous as it allows for the placement of a desired gene of interest at a specific site that is both transcriptionally active and easily amplified. Therefore, this should significantly reduce the amount of time required to isolate such high producers.
[0052]Specifically, while conventional methods for the construction of high expressing mammalian cell lines can take 6 to 9 months, the present invention allows for such clones to be isolated on average after only about 3-6 months. This is due to the fact that conventionally isolated clones typically must be subjected to at least three rounds of drug resistant gene amplification in order to reach satisfactory levels of gene expression. As the homologously produced clones are generated from a preselected site which is a high expression site, fewer rounds of amplification should be required before reaching a satisfactory level of production.
[0053]Still further, the subject invention enables the reproducible selection of high producer clones wherein the vector is integrated at low copy number, typically single copy. This is advantageous as it enhances the stability of the clones and avoids other potential adverse-side-effects associated with high copy number. As described supra, the subject homologous recombination system uses the combination of a “marker plasmid” and a “targeting plasmid” which are described in more detail below.
[0054]The “marker plasmid” which is used to mark and identify a transcriptionally hot spot will comprise at least the following sequences:
[0055](i) a region of DNA that is heterologous or unique to the genome of the mammalian cell, which functions as a source of homology, allows for homologous recombination (with a DNA contained in a second target plasmid). More specifically, the unique region of DNA (i) will generally comprise a bacterial, viral, yeast synthetic, or other DNA which is not normally present in the mammalian cell genome and which further does not comprise significant homology or sequence identity to DNA contained in the genome of the mammalian cell. Essentially, this sequence should be sufficiently different to mammalian DNA that it will not significantly recombine with the host cell genome via homologous recombination. The size of such unique DNA will generally be at least about 2 to 10 kilobases in size, or higher, more preferably at least about 10 kb, as several other investigators have noted an increased frequency of targeted recombination as the size of the homology region is increased (Capecchi, Science, 244:1288-1292 (1989)).

Problems solved by technology

While this approach has proven successful, there are a number of problems with the system because of the random nature of the integration event.
As the vast majority of mammalian DNA is in a transcriptionally inactive state, random integration methods offer no control over the transcriptional fate of the integrated DNA.
Additionally, random integration of exogenous DNA into the genome can in some instances disrupt important cellular genes, resulting in an altered phenotype.
These factors can make the generation of high expressing stable mammalian cell lines a complicated and laborious process.
Due to the translational impairment of the neo gene, transfected cells will not produce enough neo protein to survive drug selection, thereby decreasing the overall number of drug resistant colonies.
However, the success in amplification is variable.
Some transcriptionally active sites cannot be amplified and therefore the frequency and extent of amplification from a particular site is not predictable.
However, as discussed, the problem of lack of control over the integration site remains a significant concern.
However, while this type of recombination occurs at a high frequency naturally in yeast and other fungal organisms, in higher eukaryotic organisms it is an extremely rare event.
However, this particular approach is not widely applicable, because it is limited to the production of immunoglobulins in cells which endogenously express immunoglobulins, e.g., B cells and myeloma cells.
Also, expression is limited to single copy gene levels because co-amplification after homologous recombination is not included.
The method is further complicated by the fact that two separate integration events are required to produce a functional immunoglobulin: one for the light chain gene followed by one for the heavy chain gene.
However, as in the above example, expression is limited to this level because an amplifiable gene is not contegrated in this system.
Also, other researchers have reported aberrant glycosylation of recombinant proteins expressed in NS / 0 cells (for example, see Flesher et al, Biotech. and Bioeng., 48:399-407 (1995)), thereby limiting the applicability of this approach.
However, no effort was made to identify a chromosomal site from which gene expression is optimal, and as in the above example, expression is limited to single copy levels in this system.
Also, it is complicated by the fact that one needs to provide for expression of a functional recombinase enzyme in the mammalian cell.
Therefore, cells which have only undergone random integration of the vector do not survive the selection.

Method used

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  • Novel method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same
  • Novel method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same
  • Novel method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same

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example 1

Design and Preparation of Marker and Targeting Plasmid DNA Vectors

[0069]The marker plasmid herein referred to as “Desmond” was assembled from the following DNA elements:

[0070](a) Murine dihydrofolate reductase gene (DHFR), incorporated into a transcription cassette, comprising the mouse beta globin promoter 5″ to the DHFR start site, and bovine growth hormone poly adenylation signal 3″ to the stop codon. The DHFR transcriptional cassette was isolated from TCAE6, an expression vector created previously in this laboratory (Newman et al, 1992, Biotechnology, 10:1455-1460).

[0071](b) E. coli β-galactosidase gene—commercially available, obtained from Promega as pSV-b-galactosidase control vector, catalog # E1081.

[0072](c) Baculovirus DNA, commercially available, purchased from Clontech as pBAKPAK8, cat # 6145-1.

[0073](d) Cassette comprising promoter and enhancer elements from Cytomegalovirus and SV40 virus. The cassette was generated by PCR using a derivative of expression vector TCAE8 (R...

example 2

Construction of a Marked CHO Cell Line

1. Cell Culture and Transfection Procedures to Produced Marked CHO Cell Line

[0086]Marker plasmid DNA was linearized by digestion overnight at 37° C. with Bst1107I. Linearized vector was ethanol precipitated and resuspended in sterile TE to a concentration of 1 mg / ml. Linearized vector was introduced into DHFR-Chinese hamster ovary cells (CHO cells) DG44 cells (Urlaub et al, Som. Cell and Mol. Gen., 12:555-566 (1986)) by electroporation as follows.

[0087]Exponentially growing cells were harvested by centrifugation, washed once in ice cold SBS (sucrose buffered solution, 272 mM sucrose, 7 mM sodium phosphate, pH 7.4, 1 mM magnesium chloride) then resuspended in SBS to a concentration of 107 cells / ml. After a 15 minute incubation on ice, 0.4 ml of the cell suspension was mixed with 40 μg linearized DNA in a disposable electroporation cuvette. Cells were shocked using a BTX electrocell manipulator (San Diego, Calif.) set at 230 volts, 400 microfarada...

example 3

Characterization of Marked CHO Cell Lines

(a) Southern Analysis

[0088]Genomic DNA was isolated from all stably growing Desmond marked CHO cells. DNA was isolated using the Invitrogen Easy® DNA kit, according to the manufacturer's directions. Genomic DNA was then digested with HindIII overnight at 37° C., and subjected to Southern analysis using a PCR generated digoxygenin labelled probe specific to the DHFR gene. Hybridizations and washes were carried out using Boehringer Mannheim's DIG easy hyb (catalog # 1603 558) and DIG Wash and Block Buffer Set (catalog # 1585 762) according to the manufacturer's directions. DNA samples containing a single band hybridizing to the DHFR probe were assumed to be Desmond clones arising from a single cell which had integrated a single copy of the plasmid. These clones were retained for further analysis. Out of a total of 45 HisD resistant cell lines isolated, only 5 were single copy integrants. FIG. 4 shows a Southern blot containing all 5 of these si...

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Abstract

A method for achieving site specific integration of a desired DNA at a target site in a mammalian cell via homologous recombination is described. This method provides for the reproducible selection of cell lines wherein a desired DNA is integrated at a predetermined transcriptionally active site previously marked with a marker plasmid. The method is particularly suitable for the production of mammalian cell lines which secrete mammalian proteins at high levels, in particular immunoglobulins. Novel vectors and vector combinations for use in the subject cloning method are also provided.

Description

RELATED APPLICATIONS [0001]This application is a continuation-in-part of U.S. Ser. No. 08 / 819,866, filed on Mar. 14, 1997.FIELD OF THE INVENTION [0002]The present invention relates to a process of targeting the integration of a desired exogenous DNA to a specific location within the genome or a mammalian cell. More specifically, the invention describes a novel method for identifying a transcriptionally active target site (“hot spot”) in the mammalian genome, and inserting a desired DNA at this site via homologous recombination. The invention also optionally provides the ability for gene amplification of the desired DNA at this location by co-integrating an amplifiable selectable marker, e.g., DHFR, in combination with the exogenous DNA. The invention additionally describes the construction of novel vectors suitable for accomplishing the above, and further provides mammalian cell lines produced by such methods which contain a desired exogenous DNA integrated at a target hot spot.BACK...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N5/06C12N5/08C12N15/87A61K48/00C07K14/705C07K16/28C07K19/00C12N5/10C12N9/12C12N15/12C12N15/13C12N15/62C12N15/85C12N15/90G01N33/53
CPCA61K48/0008C07K14/70521C07K16/2851C07K16/2887C07K16/2896C12N2840/44C12N15/85C12N15/907C12N2800/108C12N2840/20C07K2319/02C12N15/90
Inventor REFF, MITCHELL R.BARNETT, RICHARD SPENCEMCLACHLAN, KAREN RETTA
Owner IDEC PHARM CORP
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