Assessing siRNA pharmacodynamics in a luciferase-expressing mouse

Abstract

A significant barrier to the successful general development of small-interfering RNA (siRNA) therapeutics is the ability to deliver them systemically to target organs and cell types. In this study, we have developed a mouse strain that will facilitate the evaluation of the efficacy of siRNA delivery strategies. This strain contains robust ubiquitous expression of firefly luciferase from germ line Cre-mediated recombination of the ROSA26-LSL-Luc allele. We show that luciferase is highly and uniformly expressed in all tissues examined. Using this mouse model, we describe a facile assay that enables the assessment of the pharmacodynamics of a systemically delivered siRNA formulation. These mice can also be used as universal donors, enabling the efficient and sensitive monitoring of cell trafficking or tissue transplantation. The primary advantage of this approach is that siRNA efficacy against a nonessential target can be easily evaluated in any tissue. This strain should generally enhance the ability to rapidly screen, compare and optimize various siRNA formulations for tissue-targeted or -enhanced systemic delivery in a preclinical development setting.

Figure 1. Generation of ubiquitous luciferase-expressing mice

(a) Pb-Cre4⁺, R26-LSL-Luc^fl/fl male mice were crossed with Pb-Cre4⁺, R26-LSL-Luc^fl/fl female mice to generate ubiquitous luciferase-expressing FLASH mice. Inset panel shows ovaries from both Pb-Cre4-positive and Pb-Cre4-negative mice demonstrating that Pb-Cre4 is ectopically expressed in oocytes. (b) Bioluminescence imaging (BLI) of FLASH mice demonstrates intense signal from a mouse that demonstrated germ line recombination of ROSA26-LSL-Luc (left) whereas a littermate that did not exhibit recombination showed no signal (right). Panels show ex vivo bioluminescent images of FLASH tissues. (c) Bioluminescence kinetics of intravenous (IV) versus intraperitoneal (IP) injections of substrate D-luciferin. Y-axis represents bioluminescence as photon flux (photons/s) for whole body bioluminescence values. Error bars represent mean + SEM (n = 4). (d) Luciferase expression in a panel of FLASH tissues using an in vitro luciferase assay. Values are expressed as photon flux (photons/s) per mg of protein. Error bars represent mean + SEM (n = 3). FLASH, firefly luciferase activated systemically in homozygotes; LSL, LoxP-Stop-LoxP; Sk. muscle; skeletal muscle.

Figure 2. Imaging adoptively transferred FLASH lymphocytes in vivo

(a) CD4⁺ T cells, CD8⁺ T cells, B cells, and NK cells were isolated from FLASH mice and imaged for bioluminescence intensity in vitro. Scale bar represents photons/s/cm²/sr. Average photon flux was calculated for each cell type as photons/s/cell. (b) Approximately 1 × 10⁷ CD4⁺ T cells were adoptively transferred via intravenous tail vein injection into albino C57BL6 mice (C57BL6^TYRC2J mice) and were imaged 24 hours after transfer. Focal signals of bioluminescence appear in areas that correspond to the spleen, mesenteric, superficial cervical, and inguinal lymph nodes. (c) Ex vivo bioluminescence imaging confirms the locations of CD4⁺ T-cell engraftment in various lymphoid organs. Scale bars in b and c represent photons/s/cm²/sr. FLASH, firefly luciferase activated systemically in homozygotes.

Figure 3. Liposomal small-interfering RNA (siRNA) efficacy in FLASH mice

(a) FLASH mouse embryonic fibroblasts (MEFSs) were treated with either 10, 30, 90, or 270 nmol/l of luciferase-targeted siRNA (si-Luc), 270 nmol/l of siRNA-targeting factor VII (si-FVII), or left untreated. Bioluminescence imaging (BLI) and cell viability assays were performed 72 hours after treatment. Y-axis represents bioluminescence as photon flux (photons/s) normalized to cell viability. Significant differences between si-Luc and nontreated MEFS are denoted by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean + SEM (n = 3). (b) FLASH mice were injected with either si-Luc or si-FVII and ex vivo BLI was performed on the liver and lung tissue 72 hours after injection. Scale bar represents photons/s/cm²/sr. (c) Luciferase expression levels were quantified using quantitative reverse transcriptase-PCR and an in vitro luciferase activity assay from the liver and lungs of si-Luc and si-FVII-treated FLASH mice. Y-axis represents RNA and protein levels as a percent of si-FVII-treated control mice. Error bars represent mean + SEM (n = 3). Significant differences between si-Luc- and si-FVII-treated animals are denoted by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. (d) Linear relationship between luciferase and total protein concentration from the liver of a FLASH mouse. Y-axis represents photon flux (photons/s). FLASH, firefly luciferase activated systemically in homozygotes.

Figure 4. Assessing pharmacodynamics of luciferase-targeted siRNA (si-Luc) in vivo

(a) FLASH mice were injected intravenously or intraperitoneally with si-Luc or siRNA-targeting factor VII (si-FVII) [5 mg/kg, single bolus intravenously (IV)] and tissues were analyzed for luciferase expression 72 hours after injection. (b) si-Luc efficacy evaluated in various tissues in FLASH heterozygous (fl/wt) mice 72 hours after injection. (c) Comparison of si-Luc efficacy in male and female FLASH mice 72 hours after injection. (d) Time course of si-Luc efficacy in FLASH mice. Y-axes in a–d represent photon flux (photons/s) of si-Luc treatment as a percent of si-FVII control. Significant differences between si-Luc- and si-FVII-treated animals are denoted by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. Error bars for a–d represent mean + SEM (n = 3). FLASH, firefly luciferase activated systemically in homozygotes; Sk. muscle; skeletal muscle.