Merging molecular imaging and RNA interference: early experience in live animals

Abstract

The rapid development of non-invasive imaging techniques and imaging reporters coincided with the enthusiastic response that the introduction of RNA interference (RNAi) techniques created in the research community. Imaging in experimental animals provides quantitative or semi-quantitative information regarding the biodistribution of small interfering RNAs and the levels of gene interference (i.e., knockdown of the target mRNA) in living animals. In this review we give a brief summary of the first imaging findings that have potential for accelerating the development and testing of new approaches that explore RNAi as a method for achieving loss-of-function effects in vivo and as a promising therapeutic tool.

Figure 1

Simplified RNAi pathways in mammalian cell (adapted from (Zamore and Haley, 2005)). Exogenous RNAi that are most commonly used for gene silencing in vivo are highlighted in red. Both miRNA and shRNA expression can be transcribed from exogenous DNA vector molecules that need to reach the nucleus. Micro RNA precursors are processed by Drosha RNAse complex, shRNA do not require the processing. The resultant small hairpin RNAs are exported in the cytoplasm and processed further by Dicer RNAse complex. Association with Argonaute-2 (Ago-2) exonuclease of RISC (RNA-induced silencing complex) results in antisense-guided interaction with target mRNA and a formation of silencing complex in specialized cytoplasmic bodies where target mRNA undergoes decay.

Figure 2

A- Multimodality in vivo imaging of siRNA nanoparticle delivery and RNAi effect using micro-PET/CT and bioluminescence imaging. Top row- fused micro-PET/CT images showing tumor-associated (arrow) activity one day after injection of targeted (Tf) and nontargeted (PEG) nanoparticles containing ⁶⁴Cu-DOTA-siRNA. Bottom row- luminescence images of the same mice shown above before injection and one day after injection. Graph - relative change in luciferase expression one day after injection of Tf-targeted (Tf) and nontargeted (PEG) nanoparticles containing ⁶⁴Cu-DOTA-siRNA for simultaneous PET imaging. Reprinted with permission from (Bartlett et al., 2007). B- Luminescence imaging of firefly luciferase expression knock-down by specific shRNA expression, which was co-delivered by pressure (bolus) intravenous co- injection of MDR1-luc fusion protein expression plasmid (pMDR1-Fluc, 1 µg), Renilla luciferase expression plasmid (pRLuc-N3, 1 µg; as transfection control) and the corresponding shRNA expression vector (10 µg). Bioluminescence images of Rluc expression imaged by using coelenterazine (Renilla luciferase substrate, (top) and P-glycoprotein-FLuc expression with D-luciferin (firefly luciferase substrate, bottom). Mice co-injected with 10-fold excess of control (left), scrambled shRNAi (middle), or shRNAi against MDR1 (right). Reprinted with permission from (Pichler et al., 2005). C- In vivo near-infrared optical imaging of mice that had bilaterally implanted engineered rat glioma tumors (9L-GFP and 9L-RFP) before and 48 h after intravenous injection of siRNA complexes with positively charged iron oxide nanoparticles. siRNA was designed to knock-down GFP expression (phGFP-S65T nucleotides 122–141: 5’-GCA AGC TGA CCC TGA AGT TC-3’) but not was not inhibiting RFP expression. Imaging showed a marked decrease in 9L-GFP fluorescence (P=0.0083) but not in 9L-RFP fluorescence levels. To generate GFP/RFP reconstructions, GFP and RFP images were acquired separately and then merged. Reprinted with permission from (Medarova et al., 2007). D- Imaging of luciferase activity in mice that were implanted with U251-HRE glioma cells in the flank. The cells expressed luc under control of HRE (hypoxia-responsive element). Mice were imaged on day15 of thrice weekly intratumoral injection time course. Mice on the left were injected with siRNA1589 (designed to knock-down HIF-1alpha), whereas those on the right were injected with the scrambled siRNA control. The graph quantifying the average radiance (normalized) of the glioma (U251-HRE) tumor cells imaged during the siRNA injection time course is shown below. Day 0 is the baseline luciferase activity before siRNA injection. The luciferase activity is significantly less in the group injected with siRNA1589 by day15 (*P = 0.037, n = 5 mice/group). Reprinted with permission from (Gillespie et al., 2007). E- Bioluminescence imaging of synergistic effect of siRNA and shRNA in mice. Mice were dosed in triplicate with 0.1 µg of pGL3 and either 1 µg of pSEAP (a control irrelevant plasmid), pShagLuc (shRNA expression vector), siLuc1 (siRNA construct), control, or 0.5 µg of pShagLuc and siLuc1. The imaging signals (Y-axis) were translated in to mass units of expressed enzyme (YY-axis) and demonstrate a statistically significant synergistic effect for the combined dose siLuc1 and pShagLuc in vivo. P<0.05 relative to pGL3+pSEAP and p<0.05 relative to pGL3+pShagLuc. Reprinted with permission from (McAnuff et al., 2007).