AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i

Abstract

RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo.

Figure 1

In vitro knockdown of luciferase by shRNA and U1i constructs. (a) Increasing amounts (1–250 ng) of shRNA constructs targeting firefly luciferase (shLuc1 and shLuc2) were co-transfected with 2.5 ng firefly luciferase and 0.5 ng renilla luciferase plasmid in 96-well plates containing approximately 2.5 × 10⁴ HEK293T cells. The total amount of transfected DNA was kept constant by adding the pSuper cloning vector. At 2 days after transfection, cells were lysed and firefly and renilla luciferase signals were measured. Relative luciferase activity was calculated as the ratio of firefly and renilla luciferase and plotted relative to the 100% control transfected with 250 ng pSuper. shLuc1 significantly reduced luciferase by 85%, whereas shLuc2 was inactive. Control shRNA plasmids were shGFP and shApoB. (b) Co-transfection of 200 ng U1i constructs targeting firefly luciferase (L1-L8) with firefly luciferase and renilla luciferase plasmid. Cells transfected with 200 ng control plasmid U1C were set at 100%. L4 and L5 mildly reduced luciferase expression by 30 and 40%, respectively, compared with U1C. (c) Co-transfection of 100 ng L4 and 100 ng L5 significantly improved luciferase inhibition, compared with transfection with 200 ng L4 or L5 alone (P<0.05). Data are presented as mean of three technical replicates±s.d.

Figure 2

Combination of RNAi and U1i increases luciferase knockdown in vitro. (a) Co-transfection of 50 ng control shApoB construct or 50 ng shLuc1 construct with 200 ng (or 100+100 ng) U1i plasmid and firefly luciferase and renilla luciferase plasmid. Transfections and analysis were carried out as described in Figure 1a. Cells transfected with a combination of 50 ng shApoB and 200 ng U1C were set at 100%. Co-transfection of shApoB with L4 and L5 or L4/L5 significantly reduced luciferase activity by ∼65% compared with the 100% control (P<0.05). Transfection of shLuc with U1C resulted in a significant 86% knockdown (P<0.001). (b) Co-transfection of 50 ng shLuc1 construct with 200 ng (or 100+100 ng) U1i plasmid and firefly luciferase and renilla luciferase. Note the different scales on the y axes in (a, b). Co-transfection of shLuc1 with L4 and L5 maximally reduced luciferase expression by 95%. Control U1i constructs were U1C, and H1 and H4 that target the human huntingtin sequence. Luciferase activity after co-transfection of shLuc1 with L4 and L5 or L4/L5 was significantly different from co-transfection of shLuc1 with U1C (P<0.05), and from co-transfection of shLuc1 with H1 and H4 (P<0.001) or H1/H4 (P<0.05). Data are presented as mean of three independent experiments +s.d., analyzed using factor correction.

Figure 3

Hydrodynamic delivery of shRNA and U1i constructs to murine liver inhibits luciferase activity in vivo. Mice were hydrodynamically co-transfected with 10 μg shRNA (shLuc1 or control shApoB) and 40 μg double U1i (L4/L5 or control H1/H4) plasmid and with 2 μg firefly luciferase reporter and 0.5 μg secreted alkaline phophatase (SEAP) plasmid. Both the firefly luciferase and SEAP reporter gene were expressed under control of the liver-specific AAT promoter, as hydrodynamically delivered plasmid DNA localizes mainly to the liver. Two days after transfection, bioluminescence was measured in the IVIS, and luciferase signal was calculated relative to plasma SEAP levels. Murine liver co-transfected with control constructs shApoB and H1/H4 was set at 100%. Co-transfection of shLuc1 with L4/L5 maximally reduced luciferase activity by 84%. However, luciferase knockdown was not significantly different between treatments. Data are presented as mean (n=4–8)±s.e.

Figure 4

Co-expression of RNAi and U1i from one vector backbone suppresses luciferase activity in vitro. Increasing amounts (25–250 ng) of shRNA/U1i plasmid expressing both shRNA and double U1i constructs were co-transfected with firefly luciferase and renilla luciferase plasmid and analyzed as described in Figure 1a. The total amount of transfected DNA was kept constant by adding the pro-AAV cloning vector. Cells transfected with 250 ng pro-AAV vector were set at 100%. The control vector AH expressed shApoB and double H1/H4. The U1i vector AL expressed shApoB and double L4/L5. The shRNA vector LH expressed shLuc1 and double H1/H4. The shRNA and U1i combination vector LL expressed shLuc1 and double L4/L5. AL, LH, and LL significantly reduced luciferase activity compared with the AH control vector (P<0.001) and LL was most effective and led to 85% luciferase knockdown. Data are presented as mean of three independent experiments±s.d., analyzed using factor correction.

Figure 5

AAV-mediated in vivo knockdown of luciferase using RNAi and U1i. Murine muscle was co-transduced with 1 × 10¹⁰ gc AAV-Luc and 4.5 × 10¹⁰ gc AAV-shRNA/U1i per hind limb. The gastrocnemic and adductor muscles of both hind limbs were injected. Six mice per treatment were injected, resulting in n=12. Luciferase expression was measured in the IVIS at 1, 2, 3, 4, 6, and 8 weeks post-transduction. The control vector AAV-AH expressed shApoB and double H1/H4. The U1i vector AAV-AL expressed shApoB and double L4/L5. The shRNA vector AAV-LH expressed shLuc1 and double H1/H4. The shRNA and U1i combination vector AAV-LL expressed shLuc1 and double L4/L5, and this viral vector significantly reduced luciferase expression on average by 25% during the course of the experiment (P<0.05). Data are presented as mean (n=12)±s.e.