Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Regulatory Nucleic Acid Elements

a technology of nucleic acid elements and regulatory nucleic acid, which is applied in the direction of viruses/bacteriophages, drug compositions, peptide sources, etc., can solve the problem that the introduction of te elements cannot achieve the effect of increasing productivity, canceling out chromosomal positional effects, and increasing the transcription or expression of genes

Inactive Publication Date: 2008-05-29
BOEHRINGER INGELHEIM PHARM KG
View PDF1 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to a nucleic acid sequence (SEQ ID No. 1) and its use in stable transfection of host cells. This nucleic acid sequence, known as a "TE element," has been found to increase the transcription or expression of a gene of interest in stably transfected cells. Surprisingly, the use of this element does not require the addition of any other cis-active elements, such as promoters or enhancers, and can lead to higher productivity in transfection experiments. The invention also includes expression vectors containing the TE element and methods for using them in the production of high-producing cell lines. The use of the TE element can reduce the time and costs associated with identifying high-producing cells. The invention can be applied in the development of high-producing cell lines for various applications, such as biopharmaceutical manufacturing and gene therapy.

Problems solved by technology

In transient transfections of CHO-DG44 cell pools, on the other hand, no increase in productivity can be achieved by introducing the TE elements.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Regulatory Nucleic Acid Elements
  • Regulatory Nucleic Acid Elements
  • Regulatory Nucleic Acid Elements

Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation and Cloning of the TE Element TE-A

[0319]Starting from the sequence from the hamster genome described in WO97 / 15664, which comprises, in addition to the coding region for the ubiquitin / S27a gene, adjacent 5′UTR regions including the Ub / S27a-promoter, hitherto unknown sequence regions were isolated further upstream. For this, adapter-ligated genomic CHO-DG44 DNA was used as the matrix for “nested PCRs”. The first PCR was carried out with a combination of primers with complementarity to the adapter or to a hamster sequence in the 5 region of the sequence listed in WO97 / 15664 under SEQ ID No. 5 (primer Ub20: 5′-CTCCACACATTTACACATGGACAC-3 (SEQ ID No. 39)); corresponds to nucleotides 62 to 85 (complementary sequence) of SEQ ID No. 5 from WO 97 / 15664). Then a second PCR was carried out with a second primer combination, consisting of an inner adapter primer and an inner (“nested”) hamster-specific primer (primer Ub21: 5′-GGGTTTCTCTGTGTAATAGCCATG-3 (SEQ ID No. 40); corresponds to n...

example 2

Generation of Diverse TE Expression Vectors

[0324]Starting from the 3.8 kb TE-element TE-A (FIG. 3, Sequence ID No. 1) from the CHO genome various fragments were produced by PCR which had deletions at either the 5′ or 3′-end, compared with the TE element TE-A (FIG. 4 and FIG. 5). To synthesise these fragments combinations of direct and one reverse primer were used (FIG. 5 and FIG. 6). For cloning purposes a BamHJ cutting site was attached at the 5′-end of the fragment and a BsrGI cutting site was attached at the 3′-end of the fragment by the primers. In this way 12 TE-elements of different lengths designated TE-01 to TE-12 were generated (FIG. 4 and 5). After digestion with BsrGI and BamHJ these were cloned in direct orientation into the adapter region of the base plasmids pTE4 / MCP-1 (FIG. 1B) or pTE5 / MCP-1 (FIG. 2). The fragment TE-00 (SEQ ID No. 2) was isolated from a subclone of TE-A by SacII-restriction enzyme digestion and cloned into the base vectors pBING-LC (FIG. 1A) or pBID-...

example 3

Influence of the TE Element Variant TE-00 on the Expression of GFP and Immunoglobulin G1 (IgG1)

[0325]The effect of the TE element TE-00 on the expression of the cytoplasmically located GFPs (green fluorescent protein) and a secreted monoclonal IgG1-antibody was investigated in two independent stable transfection series with CHO-DG44 cells. For this, CHO-DG44 cells were co-transfected with the following plasmid combinations or plasmid variants: control plasmids pBING-LC (FIG. 1A) and pBID-HC (FIG. 1A) without TE element pBING-LC and pBID-HC with a TE element TE-00 integrated upstream from the promoter / enhancer in direct orientation

[0326]pBING-LC and pBID-HC with a TE element TE-00 integrated upstream from the promoter / enhancer in reverse orientation

[0327]In transfection series A four pools were produced, in transfection series B ten pools were produced per variant. Equimolar amounts of the two plasmids were used. In order to arrive at the same total number of molecules, the total amo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
concentrationsaaaaaaaaaa
volumeaaaaaaaaaa
concentrationsaaaaaaaaaa
Login to View More

Abstract

The invention relates to DNA-sequences, especially transcription- or expression-enhancing elements (TE elements) and their use on an expression vector in conjunction with an enhancer, a promoter, a product gene and a selectable marker.The invention describes Sequence No. 1 and TE elements TE-01, -02, -03, -04, -06, -07, -08, -10, -11 or -12. Because of their small size, TE-06, TE-07 or TE-08 are particularly preferred. Sequence No. 1 originates from a sequence region located upstream from the coding region of the Ub / S27a gene from CHO cells.TE elements bring about an increase in the expression of the product gene, particularly when stably integrated in the eukaryotic genome, preferably the CHO-DG44 genome. Chromosomal positional effects are thereby overcome, shielded or cancelled out. In this way the proportion of high producers in a transfection mixture and also the absolute expression level are increased up to seven-fold.

Description

[0001]This application claims priority benefit from European application EP 06 117 862.0, filed July 26, 2006, which is incorporated herein in its entirety.FIELD OF THE INVENTION[0002]The invention relates to cis-active nucleic acid sequences, so-called TE elements. The TE elements preferably originate from the CHO genome. Their use in expression vectors, for example, in stable cell populations permits at least twice as high an expression of a gene of interest in a desired chromosome locus compared with vectors previously used.BACKGROUND TO THE INVENTION[0003]Mammalian cells are the preferred host cells for the production of complex biopharmaceutical proteins as the post-translational modifications are human-compatible both functionally and from a pharmacokinetic point of view. The main relevant cells types are hydridomas, myelomas, CHO (Chinese Hamster Ovary) cells and BHK (Baby Hampster Kidney) cells. The host cells are increasingly cultivated under serum- and protein-free product...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/06C12N15/00C12N15/11C12N15/85C12N5/10
CPCC07K14/47C12N15/63C12N2830/85C12N2800/107C12N2830/46C12N15/85A61P43/00C12N15/11C12N15/67
Inventor ENENKEL, BARBARASAUTTER, KERSTIN
Owner BOEHRINGER INGELHEIM PHARM KG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com