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Tissue distribution of liposome-mediated epidermal growth factor receptor antisense gene therapy
Sufi M Thomas1, Qing Zeng, Kevin F Dyer
1Department of Otolaryngology, University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213, USA.
Abstract:
Despite the widespread use of liposome-mediated gene transfer in cancer therapy protocols, little is known about the tissue distribution of intralesionally administered DNA. We have previously shown that antisense gene therapy targeting the epidermal growth factor receptor (EGFR) inhibited tumor growth in a human head and neck squamous cell carcinoma (HNSCC) xenograft model. Further investigation demonstrated lack of systemic toxicity with intramuscular or intratumoral administration of this liposomal-DNA complex. In the present study, we compared two approaches to determine the presence of exogenous DNA in the plasma and tissues of mice treated with intramuscular injection of EGFR antisense gene therapy. PCR analysis using genomic DNA plus plasmid DNA as template was 83-fold more sensitive than PCR using a mixture of total RNA and plasmid DNA as template. With the more sensitive method (able to detect fewer than 500 molecules of EGFR antisense DNA in 1 microg of genomic DNA), foreign DNA was detected in all organs up to 1 month following a single injection. In contrast, using RNA plus plasmid DNA as template, exogenous DNA was only detected at the injection site at 1 week, and was undetectable at 1 month. Optical imaging studies demonstrated plasmid DNA only at the injection site. Although less sensitive than PCCR, Southern blot hybridization showed no evidence of integration of foreign DNA into the host genome in vitro or in vivo. These results emphasize the importance of defining the assays used to detect foreign DNA and suggest that the ability to detect intralesionally administered liposomal gene therapy, in organs distant from the injection site, is directly correlated with the sensitivity of the method employed.
Insights
Detecting liposomal gene therapy DNA in distant organs depends on assay sensitivity. Highly sensitive PCR detected exogenous DNA widely, while less sensitive methods showed limited distribution, highlighting assay importance in gene therapy research.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- Liposome-mediated gene transfer is common in cancer therapy, but DNA tissue distribution after local administration is poorly understood.
- Previous work established EGFR antisense gene therapy efficacy in head and neck squamous cell carcinoma (HNSCC) xenografts with no systemic toxicity.
- Intramuscular or intratumoral administration of liposomal-DNA complexes requires clear understanding of biodistribution.
Purpose of the Study:
- To compare different methods for detecting exogenous DNA in plasma and tissues after intramuscular injection of EGFR antisense gene therapy.
- To assess the sensitivity and reliability of PCR and optical imaging for tracking gene therapy DNA distribution.
- To investigate the potential integration of foreign DNA into the host genome.
Main Methods:
- Comparison of PCR using genomic DNA plus plasmid DNA versus PCR using total RNA plus plasmid DNA for sensitivity.
- Optical imaging to visualize plasmid DNA distribution.
- Southern blot hybridization to assess DNA integration into the host genome.
Main Results:
- Highly sensitive PCR (detecting <500 DNA molecules) revealed exogenous DNA in all organs up to 1 month post-injection.
- Less sensitive PCR (RNA template) detected DNA only at the injection site within 1 week.
- Optical imaging showed DNA exclusively at the injection site; Southern blot found no evidence of DNA integration.
Conclusions:
- The detection of intralesionally administered liposomal gene therapy in distant organs is directly correlated with the sensitivity of the detection method.
- Assay selection is critical for accurately determining the biodistribution and persistence of gene therapy agents.
- Exogenous DNA was widely distributed but did not integrate into the host genome in this model.