Productivity augmenting protein factor, novel cell lines and uses thereof
Inventors
Sandig, Volker • Jordan, Ingo • Brundke, Elisabeth
Assignees
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Publication Number
US-8815540-B2
Publication Date
2014-08-26
Expiration Date
Abstract
The present invention provides a method for preparing a non-adenoviral target virus or target proteins utilizing a potent expression cell line having stably integrated into its genome a gene encoding a specific heterologous regulator protein.
Core Innovation
The invention relates to expression cells and a heterologous regulator based on adenovirus serotype 5 pIX (protein 9), including functional variants and fusions of pIX. The pIX regulator is used in host/expression cells to increase productivity and yield of non-adenoviral target viruses and/or non-adenoviral target proteins. The document describes functional embodiments including pIX-RARA and pIX-GFP, including considerations of NLS and NES behavior for PIX/PIX-GFP constructs.
The problem being solved is that interferon-mediated inhibition can reduce virus and protein production in host cells when producing non-adenoviral targets. The document reports that pIX-positive cells exhibit reduced interferon-mediated inhibition, including effects associated with TLR3/polyI:C and interferon-beta sensitivity. In addition, the document describes stable transgene maintenance and expression behavior with observed cytoplasmic/nuclear exclusion behavior for PIX-GFP constructs.
The platform includes specific expression cell types across vertebrate hosts, including a human brain-derived NC5T11 cell and avian duck retina/somite cell types and derivatives. Deposited cell lines are disclosed, including NC5T11#34 (DSMZ ACC2744) and CR.PIX(17a11b) (DSMZ ACC2749). The document further describes culturing pIX-expressing cells with a target virus/vector and isolating the target virus/protein as part of the disclosed production approach.
Claims Coverage
The independent claim is directed to an isolated CR.PIX(17a11b) cell deposited with DSMZ under accession number DSM ACC2749. Dependent claims cover two payload categories (non-adenoviral target virus or nucleic acid vectors encoding a non-adenoviral target virus/protein) and corresponding production workflows (preparing non-adenoviral target viruses and expressing non-adenoviral target proteins).
Deposited isolated CR.PIX(17a11b) cell
An isolated CR.PIX(17a11b) cell deposited with the DSMZ under accession number DSM ACC2749.
Non-adenoviral target virus or encoding vectors in CR.PIX(17a11b) cells
The isolated CR.PIX(17a11b) cell further comprising a non-adenoviral target virus, a nucleic acid vector encoding a non-adenoviral target virus, or a nucleic acid vector encoding a non-adenoviral target protein.
Selectable range of non-adenoviral target viruses
The isolated CR.PIX(17a11b) cell includes a non-adenoviral target virus selected from wild type, mutated, or deleted viruses; cold adapted or attenuated viruses; vaccine strains; viral vectors carrying heterologous gene(s); lentivirus; poxvirus; fowlpox virus; canarypox virus; and adeno-associated virus (AAV).
Preparing a non-adenoviral target virus by culturing and isolating
A method for preparing a non-adenoviral target virus by culturing a cell containing the non-adenoviral target virus and then isolating the target virus.
Preparing a non-adenoviral target protein by vector-mediated expression and isolating
A method for preparing a non-adenoviral target protein by culturing the cell with a nucleic acid vector under conditions suitable for expressing the non-adenoviral target protein, and then isolating the non-adenoviral target protein.
Overall, the claim set is centered on a specific deposited isolated CR.PIX(17a11b) cell, extended by incorporating non-adenoviral virus or nucleic acid vectors encoding non-adenoviral target virus/protein, and by methods that prepare the non-adenoviral target virus or express and isolate the non-adenoviral target protein.
Stated Advantages
Increased productivity and yield of non-adenoviral target viruses and/or non-adenoviral target proteins.
Reduced interferon-mediated inhibition, including effects associated with TLR3/polyI:C and interferon-beta sensitivity.
Stable transgene maintenance and expression.
Documented Applications
Production of non-adenoviral target viruses by using pIX-expressing cells and isolating the target virus.
Production of non-adenoviral target proteins by using pIX-expressing cells with a nucleic acid vector and isolating the target protein.
Production/handling of attenuated poxviruses including MVA as a non-adenoviral target virus.
Production of a non-adenoviral target protein example: alpha-1-antitrypsin.
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