Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-11-16
2004-03-23
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091420, C435S320100
Reexamination Certificate
active
06709844
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention disclosed herein relates to methods and compositions for the efficient production of bacterial plasmid-based shuttle vectors free of linear plasmid-derived DNA contaminants, and particularly such vectors as might be used in DNA vaccination and gene therapy.
2. Description of the Related Art
The application of recombinant DNA technology to eukaryotic cells has come to rely on shuttle vectors, plasmids that can be readily produced in bacterial culture but include elements that function in eukaryotic cells. Generally such plasmids contain a bacterial origin of replication and a selectable marker. In most cases the origin of replication is a high copy number origin so that many copies of the plasmid can replicate simultaneously in each bacterial cell thereby providing higher yields in production. The most commonly used origins of replications include pMB1, ColE1, and pUC. These origins are closely related and can be found in high and low copy number variants. The selectable marker serves to ensure that only bacteria containing the plasmid survive in culture. The eukaryotic elements most often consist of a viral or eukaryotic promoter linked to a protein coding frame leading to production of a peptide or protein when the plasmid is transduced into an appropriate eukaryotic host cell. In other cases transcription from the promoter accomplishes expression of a viral genomic RNA, leading to production of virus particles, or an antisense RNA. Various other elements may be included depending on the functions sought and these functions may dictate the arrangement of the elements involved. However no rules have been described for the arrangement of functionally unrelated elements.
SUMMARY OF THE INVENTION
The present invention is directed to methods and compositions for avoiding the production of certain side-products to plasmid replication. When the plasmid is used in pharmaceutical products, as in gene therapy or DNA vaccination procedures, these side-products complicate production and the acquisition of regulatory approval. The diversion of replicative machinery and chemical precursors constitutes a drain on bacterial metabolism potentially reducing yield of the desired product. Moreover, the side products require characterization and very possibly removal by an additional purification step.
The side-products appear to be replication intermediates on which synthesis is terminated at particular sequences found in eukaryotic transcription and translation control elements. These side products are produced generally when the bacterial origin of replication is 5′ of, and parallel in orientation to, the eukaryotic element(s).
In one aspect of the invention the production of the side-products is eliminated by inverting the orientation of the origin of replication relative to the eukaryotic element(s). In another aspect of the invention production of the side-products is eliminated by increasing the distance between the origin of replication and the eukaryotic element(s). In yet another aspect of the invention an immunostimulatory sequence is inserted between the origin of replication and the eukaryotic element(s) so that the side products may be used as a vaccine adjuvant.
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Ketter James
Knobbe Martens Olsen & Bear LLP
MannKind Corporation
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