A Mutated Human Cytomegalovirus Expression Vector With Enhanced Ability to Express Foreign Genes
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Gfi-1 (Growth Factor Independence -1) is a zinc-finger transcriptional repressor capable of binding DNA in a sequence-specific manner. It is expressed in thymus, spleen, and testis, and in the mouse, is thought to contribute to induction and progression of hematopoietic neoplasms such as T cell lymphomas. The promoters of several proto-oncogenes and cytokines contain putative gfi-1 binding sites, supporting the hypothesis that gfi-1 may contribute to T cell activation and oncogenesis by repressing genes whose expression inhibits these processes.
In addition to the gene promoters listed above, the Major Immediate Early (MIE) promoter of the human cytomegalovirus (HCMV) contains two gfi-1 binding sites. In hematopoietic cells, HCMV gene expression is downregulated due to the presence of gfi-1 binding to its promoter sequences. Mutation of these sites results in loss of gfi-1 binding and transcriptional repression of genes regulated by the MIE promoter. In addition to the implications of gfi-1?s role in the biology of HCMV, HCMV is a commonly utilized expression vector. Mutating the gfi-1 binding sites within the viral promoter can increase levels of expression of foreign genes cloned into the vector and expressed in hematopoietic cells..
Researchers at Fox Chase Cancer Center have engineered a human cytomegalovirus (HCMV) expression vector with the ability to modulate high levels of expression of inserted genes. The vector contains mutated gfi-1 binding sites in the major immediate early (MIE) promoter, allowing for high levels of expression in hematopoietic and other cells. This vector is ideal for use in gene therapy, especially in cases of hematopoietic malignancies.
The HCMV promoter is a strong promoter active in numerous cell types. Unfortunately, this promoter is down-regulated in hematopoietic cells due to gfi-1 binding, reducing its utility for gene therapy for treatment of hematologic malignancies.
The Fox Chase mutated HCMV promoters solve that problem by being unable to bind gfi-1. Therefore, gene expression driven by these promoters is not repressed in hematopoietic cells, making this vector ideal for use in gene therapy, and DNA vaccine production.
Further, since several cytokine and proto-oncogenes appear to contain gfi-1 binding motifs, mutating their promoters would allow for construction of novel vectors that permit expression of these potentially clinically relevant proteins.
The mutated HCMV expression vector is available for commercial evaluation and licensing.
US and foreign patent applications are pending. (May 30, 2000)