Enhanced gene expression system
AssigneeBaylor College of Medicine
InventorNancy Smyth Templeton
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Abstract
The present disclosure provides an enhanced gene transcription system including a systematic method of selecting efficient promoter-enhancers, of optimizing plasmid design and increasing transcription of a cDNA of interest in transfected target cells. The present invention identifies abundantly, selectively expressed genes and creates plasmids comprising the promoters-enhancers of those genes.
Claims38
A process for selecting a promoter for inclusion in a plasmid used to transfect a target cell, the process comprising the steps of:
- identifying a transcription product in high abundance in a target cell;
- identifying a promoter associated with the transcription product;
- inserting the promoter into a gene expression plasmid construct, the plasmid construct having a therapeutic gene to be expressed;
- transfecting the target cell with the gene expression plasmid construct;
- and
- verifying gene expression of the therapeutic gene in the target cell.
A process for selecting a promoter for inclusion in a plasmid to be used in gene therapy, the process comprising the steps of:
- determining a gene expression level for a plurality of transcription products in a diseased tissue;
- selecting a transcription product in high abundance in the diseased tissue and a target cell associated with the diseased tissue;
- identifying a promoter associated with the transcription product;
- inserting the promoter into a gene expression plasmid construct, the plasmid construct having a therapeutic gene to be expressed;
- transfecting the target cell with the gene expression plasmid construct;
- and
- verifying gene expression of the therapeutic gene in the target cell.
A process for designing a plasmid for transfecting a target tissue, the process comprising the steps of:
- selecting a gene expression plasmid having an origin of replication, a multiple cloning site, a therapeutic gene, a polyadenylation signal sequence, and an antibiotic resistant gene;
- identifying a transcription product in high abundance in a cell line associated with a target tissue;
- identifying a promoter associated with the transcription product;
- inserting the promoter into the gene expression plasmid in various locations close to the therapeutic gene to form a plurality of plasmid constructs;
- transfecting the target cell line with each plasmid construct;
- measuring gene expression of the therapeutic gene in the target cell line transfected with each plasmid construct;
- selecting the plasmid constructs that provide efficient gene expression in the transfected target cell line;
- and
- verifying gene expression of the therapeutic gene from the selected plasmid constructs in the target tissue.
An expression plasmid for gene therapy in breast cancer, the plasmid comprising:
- an origin of replication gene;
- a polyadenylation site;
- an antibiotic resistant gene;
- a multiple cloning site;
- a therapeutic gene;
- a GADPH promoter;
- and
- a hypoxia enhancer.
Dependencies are inferred from the claim text.