Avian telomerase reverse transcriptase
Inventors
Erbs, Philippe • Balloul, Jean-Marc • Kapfer, Marina • Silvestre, Nathalie
Assignees
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Publication Number
US-9024003-B2
Publication Date
2015-05-05
Expiration Date
Abstract
The present invention notably relates to novel recombinant telomerase reverse transcriptases, nucleic acid molecules coding them, cells comprising said nucleic acid molecule and use of these cells for the production of substance of interest.
Core Innovation
The invention relates to an avian-optimized telomerase reverse transcriptase (TERT) system, including a recombinant cDNA nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:2. When expressed in an isolated avian cell, the nucleic acid molecule exhibits telomerase reverse transcriptase (TERT) activity. The disclosed system is directed to maintaining telomeres and enabling avian telomere maintenance through expression of TERT.
The disclosure further provides engineered avian cells and vectors in which the SEQ ID NO:2 nucleic acid is used to support stable modification of the avian cell. In particular, the content describes targeting into the avian HPRT locus using homologous recombination, with an operably linked promoter associated with expression of the TERT nucleic acid. Recombinant polypeptides and nucleic acids are described with SEQ ID identity criteria.
The content also describes selection-marker architectures for generating stable immortalized cells, including positive marker configurations with suppressible or removable components and negative or suicide markers. Marker elimination is described using I-SceI and 5-fluorocytosine. Engineered cells and immortalized cell lines are described as supporting replication or production of recombinant or defective viruses, including poxvirus such as MVA, and other virus types including adenovirus, retrovirus, herpesvirus, and alphavirus.
Claims Coverage
The relevant independent claim is centered on a recombinant cDNA nucleic acid comprising SEQ ID NO:2 and demonstrating telomerase reverse transcriptase (TERT) activity upon expression in an isolated avian cell. The dependent claims refine the independent claim with additional inventive features including specific genomic insertion into HPRT, specific avian species sourcing, additional coding sequences, and genetic elements for propagating defective viruses.
Recombinant cDNA encoding SEQ ID NO:2 with avian TERT activity
A recombinant cDNA nucleic acid molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 2, wherein expression of said nucleic acid molecule in an isolated avian cell exhibits telomerase reverse transcriptase (TERT) activity.
Genomic insertion into the HPRT gene
An isolated avian cell wherein the nucleic acid molecule is inserted into the Hypoxanthine phosphorylbosyl transferase (HPRT) gene.
Isolated avian cell from Cairina moschata
The isolated avian cell is from an animal of the Cairina moschata species.
Additional nucleic acid coding a protein of interest
The isolated avian cell further comprises a nucleic acid sequence coding a protein of interest.
Complementation cassette enabling propagation of a defective virus
The isolated avian cell further comprises a complementation cassette allowing propagation of a defective virus.
Vector selection-marker configuration with positive selection surrounded by homologous sequences
The vector further comprises a first selection marker that is a positive selection marker surrounded by the homologous sequences.
Across the independent claim and its dependents, the core claimed coverage focuses on the SEQ ID NO:2 recombinant TERT cDNA with demonstrated TERT activity in isolated avian cells, further narrowed by specific HPRT-targeted insertion, Cairina moschata cell source, optional inclusion of a protein-of-interest coding sequence, optional complementation cassette for defective virus propagation, and defined vector selection-marker configurations.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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