Cell lines and their use in encapsulated cell biodelivery
Inventors
Kusk, Philip • Wahlberg, Lars Ulrik
Assignees
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Publication Number
US-8741340-B2
Publication Date
2014-06-03
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 relates to a monoclonal cell line comprising a transposon with two terminal inverted repeats and a heterologous expression construct coding for a structural gene. The structural gene codes for either a secreted biologically active polypeptide or a polypeptide or an siRNA contributing to the generation in the cells of a biologically active secreted compound. The gene is located between the terminal inverted repeats, and the transposon is inserted into the genome using a transposase.
The monoclonal cell line maintains a high and stable expression level of the polypeptide or siRNA in the absence of selective pressure, compared to the same monoclonal cell line generated without a transposase. The disclosure emphasizes transposase-based generation of stable, high-expression human cell lines for naked and encapsulated cell biodelivery of secreted therapeutics. It also specifies that the system uses non-viral transposase insertion, with transposon terminal repeats serving as substrates for the transposase.
The document further describes encapsulation or device integration for cell biodelivery, including an immunoisolatory capsule concept with a biocompatible outer membrane and an inner core/support matrix, and mentions membrane characteristics such as polyether-sulfone (PES) membranes and MWCO. It provides therapeutic examples including neurological implantation contexts, and experimental support for transposase-driven secretion using the Sleeping Beauty (SB) system, including galanin and NGF secretion. The disclosure also characterizes preferred cell line traits such as stable long-term expression and high secretion output, and includes SB hyperactive transposase variant descriptions and example constructs.
Claims Coverage
The independent claim covers a monoclonal cell line produced by transposase-mediated genomic insertion of a transposon containing terminal inverted repeats and a heterologous expression construct, where the inserted structural gene supports secretion of a biologically active compound with high stable expression absent selective pressure. The main inventive features total four: (1) the transposon architecture with terminal inverted repeats, (2) the heterologous structural gene coding scope, (3) transposase-mediated insertion into the monoclonal cell line genome, and (4) maintenance of high stable expression without selective pressure compared to generation without transposase.
Transposon with terminal inverted repeats and heterologous expression construct
A monoclonal cell line comprising a transposon, said transposon comprising two terminal inverted repeats and a heterologous expression construct coding for a structural gene either coding for a secreted biologically active polypeptide or coding for a polypeptide or an siRNA contributing to the generation in the cells of a biologically active secreted compound.
Structural gene positioned between transposase substrates
said gene being located between said two terminal inverted repeats which are substrates for a transposase.
Transposase insertion into the monoclonal cell line genome
said transposon being inserted into the genome of said monoclonal cell line using said transposase.
High and stable expression without selective pressure
wherein said monoclonal cell line maintains a high and stable expression level of the polypeptide or siRNA of I or ii absent the presence of a selective pressure compared to the same monoclonal cell line generated without a transposase.
Overall, the claim coverage is centered on a transposon-mediated, non-viral insertion of a heterologous structural gene flanked by terminal inverted repeats, where the encoded secreted biologically active polypeptide (or polypeptide/siRNA enabling a biologically active secreted compound) is maintained at high and stable expression levels without selective pressure.
Stated Advantages
Maintains a high and stable expression level of the polypeptide or siRNA in the absence of selective pressure.
Documented Applications
Cell biodelivery of secreted therapeutics using naked and encapsulated cell approaches.
Neurological implantation contexts are described for therapeutic delivery using encapsulated cells.
In vivo and in vitro experimental support is described using Sleeping Beauty (SB) system-driven secretion (e.g., galanin and NGF), with comparative results to standard transfection.
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