Cell lines and their use in encapsulated cell biodelivery
Inventors
Kusk, Philip • Wahlberg, Lars Ulrik
Assignees
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Publication Number
US-10888526-B2
Publication Date
2021-01-12
Expiration Date
Abstract
The present invention relates to generation of cell lines expressing recombinant proteins for use in naked and encapsulated cell biodelivery of secreted therapeutic molecules. In one embodiment the cell line is human. In another aspect of the invention the transposon system is used for generating a cell line for secretion of a biologically active polypeptide.
Core Innovation
The invention provides a capsule for delivery of a secreted biologically active compound to a subject, where the capsule includes a biocompatible outer membrane and an inner core comprising monoclonal cells. The monoclonal cells include a heterologous expression construct carrying a structural gene that encodes either a secreted biologically active peptide or a polypeptide or siRNA contributing to the generation, in the cells, of a biologically active secreted compound. The structural gene is located between two inverted repeats that are substrates for a transposase or other integrases.
The invention maintains that the monoclonal cells sustain a high and stable expression level of the polypeptide or siRNA absent the presence of a selective pressure, compared to the same cells generated without a transposase. The capsule further includes constraints on capsule geometry and cell loading, including an outer membrane thickness between 2 and 200 microns and a capsule volume between 1 µL and 5 µL. The capsule contains less than 10^3 monoclonal cells.
In preferred embodiments reflected in the provided claim set, the inner core can be configured with an immunoisolatory semipermeable membrane and/or a support scaffold or foam scaffold, with reticulate foam scaffold pores that disperse cells within interconnected pores. The inverted repeats can be IR/DR sequences that act as a recognition site for a transposase. Example implementations include cultured human contact-inhibited ARPE-19 cells with Sleeping Beauty transposons carrying terminal inverted repeats flanking a heterologous expression construct encoding a secreted recombinant protein.
Claims Coverage
The independent claim defines a delivery capsule with transposase/integrase substrate inverted repeats in monoclonal cells, enabling high and stable expression of a secreted gene product without selective pressure, together with capsule dimensions and cell loading.
Monoclonal cell inner core carrying a heterologous expression construct with transposase/integrase substrate inverted repeats
The capsule comprises an inner core comprising monoclonal cells, where the monoclonal cells comprise a heterologous expression construct comprising a structural gene for a secreted biologically active peptide or for a polypeptide or siRNA contributing to a biologically active secreted compound, and wherein the gene is located between two inverted repeats that are substrates for a transposase or other integrases.
High and stable expression level absent selective pressure compared to cells generated without a transposase
The monoclonal cells maintain a high and stable expression level of the polypeptide or siRNA absent the presence of a selective pressure compared to the same cells generated without a transposase.
Capsule structural dimensions and limited monoclonal cell loading
The capsule has an outer membrane thickness between 2 and 200 microns and a capsule volume between 1 µL and 5 µL, and further wherein the capsule contains less than 10^3 monoclonal cells.
Overall, claim coverage centers on a capsule-internal monoclonal cell system in which a heterologous structural gene encoding a secreted biologically active product is positioned between transposase/integrase substrate inverted repeats, producing high and stable expression without selective pressure, while the capsule is constrained by outer membrane thickness, capsule volume, and monoclonal cell count.
Stated Advantages
Maintains a high and stable expression level of the polypeptide or siRNA absent the presence of a selective pressure compared to the same cells generated without a transposase.
Documented Applications
Capsule for delivery of a secreted biologically active compound to a subject.
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