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OwnerRichard Harbottle, Matthias Bozza +1inventors
InventorsRichard Harbottle, Matthias Bozza, James A. Williams
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The present invention relates to the field of self-replicating non-integrative episomal vertebrate expression vectors useful for in gene therapy, ex vivo cell therapy, stem cell therapy, and more particularly, for improving the expression of vector encoded antigens or therapeutic genes. Such recombinant DNA molecules are useful in biotechnology, transgenic organisms, gene therapy, stem cell therapy, therapeutic vaccination, agriculture and DNA vaccines. More specifically, relates to a polynucleotide comprising at least one promoter and an S/MAR element, wherein said S/MAR element is located downstream of said promoter and wherein the nucleic acid sequence of said S/MAR element (S/MAR sequence) comprises at least 3 sequence motifs ATTA (SEQ ID NO:1) per 100 nucleotides over a stretch of at most 200 nucleotides; the present invention further relates to a composition and to a host cell comprising said polynucleotide, and to the polynucleotide for use in medicine and for use in treating genetic disease. The present invention also relates to a kit and to a device comprising said polynucleotide, and to methods and uses related to the polynucleotide.
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 polynucleotide comprising at least one promoter and an S/MAR element, wherein said S/MAR element is located downstream of said promoter and wherein the nucleic acid sequence of said S/MAR element (S/MAR sequence) comprises at least 3 sequence motifs ATTA per 100 nucleotides over a stretch of at most 200 nucleotides, wherein said S/MAR element is flanked by a splice donor and a splice acceptor, and wherein said polynucleotide further comprises an R6K gamma replication origin with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:
The polynucleotide of any one of claims 17 to 21, wherein said S/MAR is flanked by a 5′ splice donor site and a 3′ splice acceptor site.
A covalently closed circular recombinant DNA molecule comprising:
The polynucleotide of any one of claims 17 to 22 and 24 to 33 or the recombinant DNA molecule of claim 17, wherein said splice acceptor site has at least 95% sequence identity to SEQ ID NO:
Dependencies are inferred from the claim text.