Approaches of Transcriptional Regulation

A. Prior Rationale: Universal Promoters

Several universal promoters have been utilized to attempt to maximize gene expression. The LTR, CMV, and RSV promoters were isolated from Maloney retrovirus, cytomegalovirus, and Rous sarcoma virus, respectively.

These promoter elements were used because of the universal transcriptional activation over a broad host range. This universal transcription allowed for excellent but nondiscriminatory gene transcription and subsequent transgene expression. Because of the high levels of gene expression within several DNA constructs (i.e., viruses, cosmids, plasmids, etc.), these promoters are still used daily throughout the scientific community to test hypotheses which require uniform and high-level gene transcription. These were the promoters utilized in the first wave of gene therapy clinical trials, which focused on maximal gene expression and used local injection techniques to control the region of gene expression achieved. The LTR promoter was used to control herpes simplex virus thymidine kinase (HSV-TK) expression in a retroviral vector by placing retroviral producer cells into residual brain tumors to confer TK expression to the brain tumor, which could lead to conversion of a prodrug and subsequent tumor cell death. The CMV promoter was used in a replication-deficient adenovirus to deliver p53 gene expression after intralesional delivery to patients with both lung and head and neck tumors and is still under clinical investigation. The RSV promoter was employed to express HSV-TK after intralesional delivery in patients with several different tumor types.

B. Current Rationale of Tissue-Specific Promoters

A major challenge facing gene therapy is to generate vectors capable of achieving tissue- or tumor-specific expression. Initial gene therapy strategies utilized universal promoters that demonstrated gene transfer, but were associated with toxicity associated with nonspecific gene transduction (section III.A, above). Tissue-specific promoters offer a novel approach to developing transcriptionally targeted viral vectors with enhanced potential for human gene therapy applications as described below. Several important characteristics are required to develop a tissue/tumor-specific strategy for a particular disease. Fortunately, the recent explosion in our understanding of molecular events that are present in a variety of disease processes has simplified the identification of suitable promoters. Additionally the completion of the genome project and the utilization of microarray technology have enhanced the development of tissue- or tumor-specific promoters by allowing for the identification of novel but specific molecules associated with a particular disease (e.g., cancer). The advancements in molecular cloning techniques (e.g., PCR) has allowed the investigator to extract regulatory sequences from genomic DNA and evaluate each component through site directed mutagenesis analysis in plasmid expression vectors. Additionally, the development of luciferase and green fluorescent protein as well as other quantifiable transgenes has enabled the investigator to test the tissue- or tumor-specific nature of a particular promoter.

To illustrate the concept and utility of a tissue/tumor-specific promoter five such promoters have been selected from Table I. The basic rationale for

Table I

Gene Therapy Applications Of Tissue-Specific Promoters for Transcriptional Targeting

Promoter

Tissue-Specificity

Transgene

Vector

References

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