A versatile RNA vector for delivery of coding and noncoding RNAs

Abstract

The discovery that RNA viruses, lacking any DNA intermediate, can be engineered to express both coding and noncoding RNAs suggests that this platform may have therapeutic value as a delivery vehicle. Here we illustrate that a self-replicating, noninfectious RNA, modeled on influenza virus, provides one such example of a versatile in vivo delivery system for silencing and/or expressing a desired RNA for therapeutic purposes.

FIG 1

VLVs as delivery vehicles for coding and noncoding RNAs. (A) Schematic depicting engineered segments 4 (S4) and 8 (S8) of influenza A virus (IAV) encoding green fluorescent protein (GFP) or containing a microRNA (red). S8 encodes the miRNA from an intergenic region between nonstructural protein 1 (NS1) and the nuclear export protein (NEP) and was previously described (13). S4 expressing GFP flanked by the packaging sequence of influenza A virus HA (depicted by gray boxes) was previously described (17). To generate S4 coding for a small RNA, GFP was replaced with a primary miRNA using NheI and XhoI. (B) Small RNA Northern blot analysis of NHDF cells treated with IAV-124_S8 or VLV-124_S4 at a multiplicity of infection (MOI) of 3 and analyzed at 1 day posttreatment (dpt). U6 was used as a loading control. (C and D) NHDF were treated with VLV-GFP_S4-ctrl_S8 or VLV-GFP_S4-124_S8 at an MOI of 3, and expression of GFP (C) or miR-124 (D) was determined by fluorescence microscopy or small RNA Northern analysis at 1 dpt, respectively. M, mock treatment. (E) Small RNA Northern blot probed for miR-302, miR-367, and U6 upon VLV-302/367_S4 treatment performed as described for panel D.

FIG 2

Functional delivery of VLV-derived small RNAs. (A and B) NHDF were treated with VLV-ctrl_S4 or VLV-124_S4 at an MOI of 3, and expression of the endogenous miR-124 target polypyrimidine tract binding protein (PTB) (A) as well as miR-124 (B) was determined by Western or Northern blot analysis, respectively, at 1, 2, and 3 dpt. Actin or U6 served as a loading control.

FIG 3

Small RNA expression from VLVs can be modulated. (A) Madin-Darby canine kidney (MDCK) cells and MDCK cells stably expressing HA (HA-MDCK) or HA and NP (HA-NP-MDCK) were stained with a monoclonal Pan-H1-specific antibody and a monoclonal NP-specific antibody (BEI Resources). The secondary antibody was coupled to rhodamine red. Nuclei were stained with Hoechst 33342 (blue). (B and C) Primary lung cultures from GFP^+/− mice [C57BL/6-Tg(UBC-GFP)30Scha/J] were treated at an MOI of 5 with VLV-ctrl_S4 or VLV-amiR-GFP_S4 expressing either wild-type NP (NP_wt) or NP_93T. At 1 dpt, expression levels of amiR-GFP and endogenous miR-93 were analyzed by small RNA Northern blotting (B) and expression (expr.) levels of GFP transcripts were analyzed by standard quantitative PCR (qPCR) (C). *, P < 0.03 (as determined using a two-tailed, unpaired Student's t test). n.s., not significant. (D) GFP^+/− primary lung cultures were treated at an MOI of 5 with IAV, VLV-NP_wt-amiR-GFP_S4, or VLV-NP_93T-amiR-GFP_S4 or were mock treated (M). At 4 dpt, cell survival was determined using a Cell Titer-Glo assay (Promega) according to the manufacturer's instructions. *, P < 0.02 (as determined using a two-tailed, unpaired Student's t test). n.s., not significant. (E) GFP^+/− primary lung cultures were treated at an MOI of 5 with VLV-NP_wt or VLV-NP_93T expressing either a scrambled sequence (ctrl_S4) or amiR-GFP. At 4 dpt, GFP expression was analyzed by WB. Expression levels of NP and actin are shown.

FIG 4

VLV-delivered small RNAs result in efficient target gene knockdown in hematopoietic cells. (A) GFP^+/− BMMs from GFP^+/− mice [C57BL/6-Tg(UBC-GFP)30Scha/J] were treated at two different MOIs with VLV-124_S4, and expression of miR-124 was analyzed by small RNA Northern blotting at 1 dpt. (B) GFP^+/− BMMs were treated at an MOI of 5 with VLV-ctrl_S4 or VLV-amiR-GFP_S4. At 1 dpt, expression levels of GFP transcripts were analyzed by standard qPCR. *, P < 0.002 (as determined using a two-tailed, unpaired Student's t test). (C) GFP^+/− BMMs were treated at an MOI of 5 with VLV-ctrl_S4 or VLV-amiR-GFP_S4. At 3 dpt, expression of GFP protein was determined by FACS analysis using intracellular staining with a NP-specific monoclonal antibody (BEI Resources, 1:500) and BD Cytofix and BD Perm/Wash according to the manufacturer's recommendation. The histogram shows GFP expression of NP⁺ BMMs. (D) C57BL/6 mice were intranasally treated with 10⁸ PFU of VLV-ctrl_S4 or VLV-124_S4. RNA from whole lungs was isolated at 1 dpt, and expression of miR-124 was determined by small RNA Northern analysis.