Cell culture methods involving HDAC inhibitors or rep proteins
Inventors
Tiernan, Aubrey R. • TIPPER, Christopher
Assignees
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Publication Number
US-11898170-B2
Publication Date
2024-02-13
Expiration Date
Abstract
The invention relates to methods of culturing cells, generating cell lines, and delivering polynucleotides to cells involving the use of HDAC inhibitors and/or adeno-associated virus (AAV) rep proteins.
Core Innovation
The disclosed invention relates to developing a stable cell line comprising a recombinant AAV (rAAV) vector sequence stably integrated into chromosome 19 for producing rAAV. The method provides a rAAV vector sequence together with a rep mRNA selected from rep68 mRNA, rep78 mRNA, or a combination thereof to a HeLa cell culture, and the culturing conditions allow integration of the rAAV vector into the HeLa cell genome.
The method includes identifying a HeLa cell that contains the rAAV vector sequence integrated into chromosome 19 of the HeLa cell genome. A HeLa cell line is then generated by culturing the mammalian cell under conditions that allow for generation of a cell line that produces rAAV, and the production outcome is characterized by rAAV titer from the resulting stable cell line.
The disclosed examples and summary describe co-delivering rAAV vectors with AAV rep68/rep78 mRNA in HeLa S3 to improve stable clone generation and rAAV production. The document further describes optional use of HDAC inhibitors, including trichostatin A (TSA), during or in relation to co-delivery to support enhanced outcomes, and integration assessment approaches are described using PCR, FISH, and/or FACS/selection.
Claims Coverage
The document includes one independent claim covering a method to develop a stable HeLa cell line with chromosome 19 integration for producing rAAV, with an rAAV titer threshold after at least four days. The independent claim contains three inventive features that specify the rep mRNA selection, chromosome 19 integration and identification of integrated cells, and generation of an rAAV-producing stable cell line with a production titer requirement.
Chromosome 19-stable integration in a stable HeLa cell line for rAAV production
A method of developing a stable cell line comprising a recombinant AAV (rAAV) vector sequence stably integrated into chromosome 19 for producing rAAV, including culturing the HeLa cell culture under conditions that allow for integration and identifying a HeLa cell containing the rAAV vector sequence integrated into chromosome 19 of the HeLa cell genome, followed by culturing to generate a cell line that produces rAAV.
Providing rAAV vector with selected rep mRNA (rep68 and/or rep78) to HeLa cells
Providing a rAAV vector comprising the rAAV vector sequence and a rep mRNA selected from the group consisting of rep68 mRNA, rep78 mRNA, and a combination thereof to a HeLa cell culture.
Stable-cell-line rAAV titer threshold after at least four days
Producing rAAV by the generated cell line at a titer of at least 8.5×10^10 GC/ml when the HeLa cell culture is cultured for at least four days.
Overall claim coverage is centered on developing a stable HeLa cell line in which an rAAV vector sequence is integrated into chromosome 19, using co-provision of an rAAV vector sequence together with selected rep68 and/or rep78 mRNA, identifying integrated cells, and culturing to generate an rAAV-producing stable line with an rAAV titer threshold measured as GC/ml after at least four days.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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