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Promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression
Abstract:
One of the major limitations to current gene therapy is the low-level and transient vector gene expression due to poorly defined mechanisms, possibly including promoter attenuation or extinction. Because the application of gene therapy vectors in vivo induces cytokine production through specific or nonspecific immune responses, we hypothesized that cytokine-mediated signals may alter vector gene expression. Our data indicate that the cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) inhibit transgene expression from certain widely used viral promoters/enhancers (cytomegalovirus, Rous sarcoma virus, simian virus 40, Moloney murine leukemia virus long terminal repeat) delivered by adenoviral, retroviral or plasmid vectors in vitro. A constitutive cellular promoter (beta-actin) is less sensitive to these cytokine effects. Inhibition is at the mRNA level and cytokines do not cause vector DNA degradation, inhibit total cellular protein synthesis, or kill infected/transfected cells. Administration of neutralizing anti-IFN-gamma monoclonal antibody results in enhanced transgene expression in vivo. Thus, standard gene therapy vectors in current use may be improved by altering cytokine-responsive regulatory elements. Determination of the mechanisms involved in cytokine-regulated vector gene expression may improve the understanding of the cellular disposition of vectors for gene transfer and gene therapy.
Insights
Cytokines like interferon-gamma and tumor necrosis factor-alpha can reduce gene therapy vector expression. Modifying cytokine-responsive elements in vectors can enhance gene transfer and therapy effectiveness.
Area of Science:
- Gene Therapy
- Molecular Biology
- Immunology
Background:
- Gene therapy faces limitations due to low-level and transient vector gene expression.
- Immune responses, including cytokine production, may impact vector performance.
- Mechanisms of promoter attenuation and extinction are not fully understood.
Purpose of the Study:
- To investigate the hypothesis that cytokine-mediated signals alter vector gene expression.
- To identify specific cytokines that inhibit transgene expression from common viral promoters.
- To explore strategies for improving gene therapy vector efficacy by modulating cytokine responses.
Main Methods:
- In vitro studies using adenoviral, retroviral, and plasmid vectors.
- Exposure of cells to cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha).
- Analysis of transgene expression levels, mRNA stability, and cellular effects.
- In vivo studies involving administration of neutralizing anti-IFN-gamma monoclonal antibody.
Main Results:
- IFN-gamma and TNF-alpha significantly inhibit transgene expression from common viral promoters (CMV, RSV, SV40, MMLV LTR).
- A constitutive cellular promoter (beta-actin) demonstrated less sensitivity to cytokine-induced inhibition.
- Inhibition occurs at the mRNA level; cytokines do not degrade vector DNA or affect overall protein synthesis.
- Administration of anti-IFN-gamma antibodies enhanced transgene expression in vivo.
Conclusions:
- Cytokine-mediated inhibition is a significant factor affecting gene therapy vector expression.
- Viral promoters/enhancers are susceptible to inhibition by IFN-gamma and TNF-alpha.
- Gene therapy vectors can be improved by redesigning cytokine-responsive regulatory elements.
- Understanding cytokine-regulated vector expression is crucial for advancing gene transfer and therapy.