Non-integrating DNA vectors for the genetic modification of cells
AssigneeDeutsches Krebsforschungszentrum
InventorsJames A. Williams, Matthias Bozza, Richard Harbottle
Life of this patent
Estimating the term…
Abstract
The present invention relates to a polynucleotide comprising at least one promoter and an S/MAR element, wherein the S/MAR element is located downstream of the promoter in the 3′ UTR of the transcription unit and wherein the S/MAR element is flanked by a 5′ splice donor site and a 3′ splice acceptor site; the present invention further relates to a composition comprising the polynucleotide, and to the polynucleotide for use in medicine and for use in treating genetic disease.
Claims27
A method for improving the expression and establishment efficiency of a self-replicating non-integrative episomal S/MAR expression vector in a target vertebrate cell comprising the following steps:
- a. providing an episomal S/MAR expression vector comprising:
- i. a bacterial replication-selection region comprising a bacterial origin of replication and a selectable marker gene;
- ii. a transcription unit for expression of a transgene in a vertebrate cell, comprising a promoter, a 5′ UTR, a transgene, and a 3′ UTR;
- iii. an S/MAR insert located within said 3′ UTR;
- and
- b. modifying the episomal S/MAR expression vector such that the S/MAR is flanked by a 5′ splice donor site and a 3′ splice acceptor site within said 3′ UTR, whereby the resultant self-replicating non-integrative episomal S/MAR expression vector has improved expression and establishment efficiency after transfection of a vertebrate cell.
A covalently closed circular recombinant DNA molecule comprising:
- a. a transcription unit for expression of a transgene in a vertebrate cell, comprising a promoter, a 5′ UTR, a transgene, and a 3′ UTR;
- b. an S/MAR located within said 3′ UTR, wherein said S/MAR is flanked by a 5′ splice donor site and a 3′ splice acceptor site;
- c. an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:
- 1, SEQ ID NO:
- 2, SEQ ID NO:
- 3, and SEQ ID NO:
- 4;
- and
- d. a functional variant of an RNA-OUT selectable marker gene comprising at least a 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:
- 5 and SEQ ID NO:
- 7, and wherein a gene product of said functional variant of the RNA-OUT selectable marker gene regulates an RNA-IN regulated selectable marker gene.
Dependencies are inferred from the claim text.