Targeting Expression with Light Using Caged DNA

Summary:

Gene therapy is one of the most promising prospects in the biomedical and bioorganic realms. However, success within in vivo gene therapy is restricted by two significant limiting factors: site-specific gene delivery and subsequent localized expression within the target cell population. Conventional molecular approaches to manipulating genes in live embryos and animals do not allow for precise up-or-down regulation of gene expression on an individual cellular basis. One novel approach to overcome these obstacles involves the use of caged compounds, which possess a covalently attached group that can be released upon exposure to various wavelengths of light. The bound substance deactivates the primary molecule by inducing unfavorable physical and chemical conformations until photoactivation. Upon release of the photolabile aptamer, the previously inert compound (e.g. oligonucleotides, transcription factors, and DNA/RNA-dependent enzymes) initiates specific cellular pathways for regulated gene expression.

In this report, researchers utilized caged plasmids coding for luciferase and transfected into roughly 1-cm diameter targets in the skin of rats via gold particle bombardment. They hoped to characterize the potential for light-induced expression with higher spatiotemporal control and targeting. The plasmids were rendered inert by 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE), a photoactive caging group. The effectiveness of the DMNPE cage groups is evident with unchanged luciferase expression levels and equivalent to that of nontransfected skin sites. Upon exposure to 355-nm laser light, the uncaged plasmid induces increased luciferase expression within the target sites. Progressively increasing levels of laser dosage generates proportional increases in luciferase production.

Figure 1: Effect of light on luciferase expression in rat skin. Particle-mediated transfection of caged and native pCEP-luciferase was performed on ~1-cm diameter sites in rat skin. Luciferase expression of skin sites transfected with caged plasmid is equal to levels in nontransfected skin. Exposure of skin sites transfected with caged plasmids to increasing amounts of 355-nm laser light increases expression to 6 ± 3, 12 ± 4, and 17 ± 6% of control, respectively. The right bar indicates highest expression (40 ± 12% of control) from skin sites transfected with caged plasmids exposed to light before delivery. The asterisks indicate difference from no light exposure by two-way repeated measures analysis of variance (n = 4; mean ± S.E.).

In addition, DMNPE-caged GFP (green fluorescent protein) plasmids were introduced into HeLa cells through liposome-transfection. Further analysis revealed that the presence of the DMNPE cage groups reversibly blocks gene expression. GFP expression levels remained constant between transfected and normal cells. With application of the 355-nm laser light, GFP expression is subsequently induced within the target HeLa cell populations. Parallel in vitro research confirmed the results from both cases above and suggested regulation of gene expression at the transcription level due to reduced fabrication of mRNA from the GFP plasmid.

Figure 2: Effects of light on native and caged GFP expression in HeLa cultures. HeLa cells were liposome-transfected with caged and native GFP plasmids. The expression level of native pGFP was 43 ± 4.3% of cells (n = 11, mean ± S.E.). Percentages of expression were normalized to this group. Without exposure to light (i.e. with 0 J/cm² of 365-nm light), the fraction of HeLa cells that express caged pGFP (solid bar) is 25.8% of native plasmid expression levels (n = 7, gray bars). After exposure to 0.25 or 0.5 J/cm² of light, expression of the caged material increases to 50% of control. The asterisks indicate significant difference from expression of caged plasmids that received no light exposure (p < style="">t test). Cultures transfected with caged pGFP and treated with 2.8 and 5.6 J/cm² of light showed decreasing expression levels of 20 and 10%, respectively. Native GFP expression levels also decreased with increasing post-transfection, light exposure, from a normalized 100% with no light exposure to 81, 24, and 10% with a light exposure of 0.5, 2.6, or 5.6 J/cm², respectively. Cultures exposed to 2.8 or 5.6 J/cm² of light after transfection showed significantly lower levels of expression than those that received no light (denoted by crosses, p < style="">t test). Plasmids exposed to the highest dose of light (5.6 J/cm²) before transfection (bar labeled Pre-Flash) express at levels equal to control plasmids that received no light.

Why I Chose This Paper:

Light regulated gene expression has the potential to offer unprecedented spatiotemporal control in a wide variety of applications including genetics and cancer research. Different spectra of light can penetrate the skin of the human body without toxic side effects to the surrounding tissue. As the database for available photoactive chemical aptamers and corresponding carriers becomes more firmly established, scientists may finally gain the ability to control differentiation within targeted cells (i.e. stem cells) with exact timing and activation of the correct biochemical pathways. Each year, more than 1 million American citizens are diagnosed with some form of cancer. The National Cancer Institute estimates that nearly 10.5 million people are now living with a previous diagnosis of cancer. Current technology and techniques are limited by the cancer type, location, and progression of the tumor. Devastating side effects along with unsatisfactory success rates have forced scientists into searching for remedies with minimal side effects and overcomes non-specific targeting of healthy cells. Existing technologies and treatments (i.e. radiotherapy, chemotherapy, and surgery) are often administered in combination to increase overall success rates. Possible advancements in cancer research involve nanoparticle carriers with payloads of modified oligonucleotides (i.e. siRNA) or powerful anticancer drugs attached by photoreactive linkers. Photonic excitation and ensuing release would then occur in localized regions of the cancerous tissue with minimum damage.