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Toxicogenomics of non-viral vectors for gene therapy: a microarray study of lipofectin- and oligofectamine-induced
Yadollah Omidi1, Andrew J Hollins, Mustapha Benboubetra
1Centre for Genome-based Therapeutics, Cardiff University, The Welsh School of Pharmacy, Redwood Building, King Edward VII Avenue, CF10 3XF Cardiff, UK.
Abstract:
Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.
Insights
Cationic lipid (CL) formulations used for gene delivery can alter global gene expression in cells, potentially impacting gene therapy safety. These unintended changes may affect experimental outcomes and therapeutic efficacy.
Area of Science:
- Molecular Biology
- Gene Therapy
- Cell Biology
Background:
- Cationic lipid (CL) formulations are widely used non-viral vectors for delivering genetic material.
- The impact of these CL delivery systems on global gene expression in target cells remains largely unknown.
- Understanding geno-compatibility is crucial for the safe application of CL formulations in gene-based therapies.
Purpose of the Study:
- To investigate the effects of Lipofectin and Oligofectamine on gene expression profiles in human A431 epithelial cells.
- To assess the geno-compatibility of CL formulations by examining global gene expression changes.
- To evaluate the downstream functional consequences of CL-induced gene expression alterations.
Main Methods:
- Utilized cDNA microarray expression profiling to analyze gene expression changes in A431 cells treated with Lipofectin and Oligofectamine.
- Quantified alterations in gene expression at manufacturer-recommended concentrations.
- Assessed cellular apoptosis using annexin V-FITC flow cytometry and DNA damage via single cell gel electrophoresis (COMET assay).
Main Results:
- Lipofectin and Oligofectamine treatments induced significant, dose-dependent changes in the expression of multiple genes.
- Oligofectamine altered the expression of 27 genes, while Lipofectin affected 10 genes (more than 2-fold change).
- CL treatment increased the tendency for early apoptosis, with Oligofectamine showing a greater effect than Lipofectin; no significant DNA damage was observed.
Conclusions:
- CL formulations alone can cause inadvertent gene expression changes, posing potential safety implications for gene-based therapies.
- These non-target gene expression changes may interfere with or mask desired outcomes in gene therapy and gene silencing experiments.
- Careful consideration of formulation-induced effects is necessary for accurate interpretation of experimental results and therapeutic development.
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