A Versatile RNA Vector for Delivery of Coding and Noncoding RNAs Sonja Schmid, Lum C. Zony and Benjamin R. tenOever J. Virol. 2014, 88(4):2333. DOI: 10.1128/JVI.03267-13. Published Ahead of Print 4 December 2013.
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A Versatile RNA Vector for Delivery of Coding and Noncoding RNAs Sonja Schmid, Lum C. Zony, Benjamin R. tenOever Dept. of Microbiology at the Icahn School of Medicine at Mount Sinai, New York, New York, USA
T
he capacity to deliver foreign RNA to a specific tissue of interest is a significant barrier preventing the therapeutic translation of many basic science discoveries. This is perhaps best exemplified with RNA interference (RNAi). RNAi has the capacity to repress expression of a desired transcript through recruitment of effector protein complexes by complementary small RNAs (1). The level of RNAi-mediated posttranscriptional silencing is generally determined by the amount of complementarity between the small RNA and its target. Endogenous microRNAs (miRNAs) generally repress gene expression ⬃2- to 3-fold due to imperfect complementarity to the target gene sequence (2). However, the silencing capacity of perfectly complementary small RNAs, such as engineered artificial miRNAs (amiRNAs), is significantly higher (3–5), providing a promising strategy for therapeutics. Unfortunately, efficient in vivo delivery strategies for foreign RNA are significantly limited and have thus far hindered the therapeutic potential of RNAi to transform the field of medicine (6). Although no RNA virus, void of a DNA intermediate, has been found to encode a canonical miRNA (7–9), we, and others, have shown that RNA viruses can be engineered to generate functional small RNAs in vivo (10–13). Here, we explored the potential of replicationincompetent RNA virus-like vectors (VLVs) for the production of small RNAs. We show that influenza A virus (IAV)-derived VLVs can be engineered to deliver functional small RNAs in primary cells and in vivo, leading to efficient target gene knockdown. To generate replication-incompetent VLVs capable of eliciting RNAi, we used a previously published system for production of IAV-based VLVs not encoding the viral glycoprotein hemagglutinin (HA) (14–16). All constructs used for VLV production were based on mouse-adapted IAV strain A/Puerto Rico/8/1934(H1N1) (PR8) and have been described elsewhere (17). We replaced the open reading frame (ORF) of segment 4 with either a scrambled RNA (ctrlS4) or the primary Mus musculus miR-124-2 transcript (124S4) (Fig. 1A). We produced and propagated the respective VLVs on Madin-Darby canine kidney (MDCK) cells stably expressing HA (HA-MDCK), as previously described (14– 16). HA-MDCK cells were also utilized to determine VLV titers by standard plaque assay. First, we compared the miR-124 expression levels of replication-competent IAV-expressing miR-124 (described in reference 13, depicted in Fig. 1A, and referred to as IAV-124S8 here) and the replication-incompetent vector, VLV124S4. Surprisingly, normal human dermal fibroblasts (NHDF) treated with VLV-124S4 exhibited 3-fold-higher expression of miR-124 than cells treated with IAV-124S8, as shown by small RNA Northern analysis (Fig. 1B) (18). Encouraged by this result, we next aimed to explore the maxi-
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mum expression capacity of the VLVs. In an effort to produce both coding and noncoding RNAs from a single VLV, we utilized segment 4 expressing green fluorescent protein (GFP) (Fig. 1A) in conjunction with segment 8 expressing scrambled RNA or miR124 (here referred to as VLV-GFPS4-ctrlS8 or VLV-GFPS4-124S8). NHDF treated with either VLV showed high expression of GFP at 1 day posttreatment (dpt) (Fig. 1C). Furthermore, NHDF treated with VLV-GFPS4-124S8 expressed high levels of miR-124 (Fig. 1D), demonstrating that VLVs can simultaneously deliver both coding and noncoding RNAs into primary human cells. We next determined whether VLVs could express multiple noncoding RNAs. To this end, we cloned the miR-302/367 cluster into segment 4 (19). This miRNA cluster contains five tandem hairpins for miR-302b, miR-302c, miR-302a, miR-302d, and miR-367, respectively (302/367S4). Following rescue of the influenza virus-based particles, NHDF cells were treated to assess small RNA levels. VLV-302/367S4 expressed high levels of miR-302 and miR-367 (Fig. 1E). While Northern blot analysis could not distinguish between miR-302a, -b, -c, and -d, it is noteworthy that miR302 and miR-367 could be detected. Given that these miRNAs represent the 5= and 3= ends of the noncoding RNA, these results suggest that the five miRNAs were processed fully when encoded within the VLV. Taking those results together, these experiments suggest that VLVs can be engineered to deliver multiple coding and noncoding RNAs. We next determined whether VLV-delivered miR-124 is functional. To this end, we treated NHDF with VLV-124S4 and analyzed the expression levels of the endogenous miR-124 target, polypyrimidine tract binding protein (PTB), by Western blotting (WB) (20). Following treatment with VLV-124S4, PTB levels were reduced by 75% and 60% at 2 and 3 dpt, respectively (Fig. 2A). Consistent with this result, miR-124 was robustly expressed at 1, 2, and 3 dpt (Fig. 2B). Deep sequencing of IAV-based small RNA expression systems has suggested that the vector can generate ⬃50,000 copies of a miRNA per cell in less than 6 h (10). Given that this level of expression exceeds that of the majority of cellular miRNAs, this
Received 5 November 2013 Accepted 25 November 2013 Published ahead of print 4 December 2013 Address correspondence to Benjamin R. tenOever,
[email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03267-13
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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.
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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-124S8 or VLV-124S4 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-GFPS4-ctrlS8 or VLV-GFPS4-124S8 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-302/367S4 treatment performed as described for panel D.
delivery system may result in unwanted cytotoxicity. As such, we next wanted to determine whether production of exogenous RNAs could be modulated in the context of the VLV. To this end, we utilized a previously described strategy that relies on the expression of host miRNAs to limit virus infection (14, 21–27). For this approach, the viral genome of interest is engineered to contain one or more perfectly complementary target sites for a host miRNA, resulting in cleavage of the viral target site-containing transcript by the host RNAi machinery. This ultimately leads to attenuation of viral replication when an essential gene is silenced. We utilized previously described engineered segment 5, expressing nucleoprotein (NP) but targeted by a ubiquitous host miRNA, miR-93 (NP93T) (18), to limit VLV activity. IAV containing a NP93T segment is highly attenuated in vitro and in vivo as NP expression is necessary for the viral RNA-dependent RNA polymerase (RdRp) to function (18). In an effort to produce VLVs containing NP93T, we generated a second complementing cell line expressing both HA and NP (HA-NP-MDCK) (Fig. 3A). To address whether the silencing capacity of VLVs could be modulated, we compared NPwt or NP93T VLVs that harbored an additional artificial miRNA against GFP (28) (amiR-GFPS4). Both the amiRGFP levels and silencing capacity for these VLV constructs were analyzed in primary lung cultures derived from GFP-transgenic mice. As expected, treatment with VLV-NP93T-amiR-GFPS4 led to (5-fold) reduced expression of amiR-GFP compared to VLV-
FIG 2 Functional delivery of VLV-derived small RNAs. (A and B) NHDF were treated with VLV-ctrlS4 or VLV-124S4 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.
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NPwt-amiR-GFPS4 (Fig. 3B), resulting in small RNA levels that were comparable to those of endogenous miRNAs. Excitingly, despite reduced amiRNA expression, both VLVs led to comparable levels of knockdown of GFP transcripts at 1 dpt (Fig. 3C). We next used a commercially available cell viability assay to monitor cytotoxicity in VLV-treated primary lung cultures. These data demonstrated that, while VLV-NPwt resulted in approximately 50% cell survival, reduced NP expression (VLV-NP93T) was sufficient to yield a vector that showed no significant difference from mocktreated cells in cytotoxicity (Fig. 3D). Furthermore, at 4 dpt, we observed a 42% knockdown of GFP protein in cells treated with either VLV-NPwt-amiR-GFPS4 or VLV-NP93T-amiR-GFPS4 (Fig. 3E). As expected, treatment with VLV-NP93T resulted in reduced expression of NP compared to that seen with VLV-NPwt (Fig. 3E). Taken together, these data show that VLVs can be used to deliver amiRNAs into primary cells at a desired concentration, leading to efficient transient silencing of a target gene. Furthermore, the newly generated HA-NP-MDCK cells increase the versatility of the VLVs by allowing the production of vectors that express a targeted NP. While we used a previously described NP that has two target sites for a ubiquitously expressed mammalian miRNA, this technology could also be applied to cell- or tissue-specific miRNAs (7). In addition, small-RNA expression levels can be regulated by modulating the loop size and processing efficiency of a given amiRNA (29). We next tested whether the VLVs would allow target gene knockdown in hematopoietic cells. We chose bone marrow-derived macrophages (BMM), since resident macrophages in the lung would be likely to take up VLVs upon intranasal treatment. Small RNA Northern blot analysis showed expression of miR-124 at 1 dpt with VLV-124S4 (Fig. 4A). Furthermore, GFP transcripts were reduced by approximately 40% at 1 dpt with VLV-amiRGFPS4 (Fig. 4B). To analyze GFP protein expression, we stained BMMs treated with either VLV-ctrlS4 or VLV-amiR-GFPS4 with an antibody detecting NP and performed fluorescence-activated cell sorter (FACS) analysis. As shown in Fig. 4C, treatment with VLV-amiR-GFPS4 reduced expression of GFP in NP⫹ cells at 3 dpt. Finally, we tested whether the VLVs could deliver a small RNA in vivo. To this end, we treated wild-type mice intranasally
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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)
Influenza Virus-Based Vectors for the Delivery of RNAs
FIG 4 VLV-delivered small RNAs result in efficient target gene knockdown in
hematopoietic cells. (A) GFP⫹/⫺ BMMs from GFP⫹/⫺ mice [C57BL/6-Tg(UBCGFP)30Scha/J] were treated at two different MOIs with VLV-124S4, 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-ctrlS4 or VLV-amiR-GFPS4. 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-ctrlS4 or VLV-amiR-GFPS4. 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-ctrlS4 or VLV-124S4. RNA from whole lungs was isolated at 1 dpt, and expression of miR-124 was determined by small RNA Northern analysis.
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with VLV-124S4. Excitingly, we could detect robust levels of miR124 in whole lungs at 1 dpt (Fig. 4D). While future studies focused on demonstrating in vivo silencing with VLVs will be required, these data suggest that this system may be an exceptional means of respiratory tract delivery for siRNAs. We show here for the first time that replication-incompetent VLVs lacking a DNA intermediate can be used to deliver small RNAs. Target gene knockdown in primary human and murine fibroblasts as well as hematopoietic cells could be observed for at least 3 dpt. Furthermore, we show that miRNA-mediated silencing of the vector represents a strategy that permits one to modulate small RNA production and eliminate cellular toxicity while also defining tissue specificity if desired. In addition, these VLVs can be engineered to simultaneously express both coding and noncoding RNAs of interest. Taking the data together, we show that RNA virus-based VLVs are versatile vectors that could be utilized for a range of genetic applications. Vectors based on RNA viruses that lack a DNA intermediate possess a number of desirable attributes (10, 30). In contrast to vectors based on DNA viruses or lentiviruses, VLVs do not integrate into the host genome and would therefore not perturb endogenous gene expression. Furthermore, these vectors appear to bypass many of the cellular toxicities that have thus far been documented for stably integrating vectors as long-term exogenous expression of small RNAs saturates the endogenous RNAi pathway (31, 32). Furthermore, IAV vectors with limited replicative capacity have already been FDA approved, suggesting this platform to be safe (33). In all, this current report provides an alternative methodology for the delivery of both coding and noncoding RNAs and demonstrates the versatility of this vector-based delivery platform for future utilities.
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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-ctrlS4 or VLV-amiR-GFPS4 expressing either wild-type NP (NPwt) or NP93T. 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-NPwt-amiR-GFPS4, or VLV-NP93T-amiR-GFPS4 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-NPwt or VLV-NP93T expressing either a scrambled sequence (ctrlS4) or amiR-GFP. At 4 dpt, GFP expression was analyzed by WB. Expression levels of NP and actin are shown.
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ACKNOWLEDGMENTS This research was sponsored in part by a grant from the National Eye Institute (1R01EY023287-01). Furthermore, the following reagent was obtained through BEI Resources, NIAID, NIH: Monoclonal Anti-Influenza A Virus NP, Clone IC5-1B7 (produced in vitro), NR-4544. The Pan-H1-specific monoclonal antibody was a kind gift of Peter Palese (Icahn School of Medicine at Mount Sinai, New York).
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