Bicistronic gene transfer tools for delivery of miRNAs and protein coding sequences

Abstract

MicroRNAs (miRNAs) are a category of small RNAs that modulate levels of proteins via post-transcriptional inhibition. Currently, a standard strategy to overexpress miRNAs is as mature miRNA duplexes, although this method is cumbersome if multiple miRNAs need to be delivered. Many of these miRNAs are found within introns and processed through the RNA polymerase II pathway. We have designed a vector to exploit this naturally-occurring intronic pathway to deliver the three members of the sensory-specific miR-183 family from an artificial intron. In one version of the vector, the downstream exon encodes the reporter (GFP) while another version encodes a fusion protein created between the transcription factor Atoh1 and the hemaglutinin epitope, to distinguish it from endogenous Atoh1. In vitro analysis shows that the miRNAs contained within the artificial intron are processed and bind to their targets with specificity. The genes downstream are successfully translated into protein and identifiable through immunofluorescence. More importantly, Atoh1 is proven functional through in vitro assays. These results suggest that this cassette allows expression of miRNAs and proteins simultaneously, which provides the opportunity for joint delivery of specific translational repressors (miRNA) and possibly transcriptional activators (transcription factors). This ability is attractive for future gene therapy use.

Figure 1

Bifunctional vector design and processing of transcripts. The vector consists of the EF1α promoter that will drive expression of the miR-183 family of genes from the intron designated by the splice donor (SD) and splice acceptor (SA) site, and an exon encoding Atoh1 fused to the hemagluttinin influenza epitope (HA). Once the plasmid is transcribed into RNA, endogenous enzymes present in transfected cells should recognize the SD and SA sites to release the intron containing the primary miRNA transcript (A); clip it into the three distinct pre-miRNAs, export them from the nucleus (B); and then further process them into mature miRNAs (C). As the miRNAs follow their own maturation pathway, the spliced Atoh1-HA-encoding polyA+ transcript is exported from the nucleus and processed as mRNA.

Figure 2

Content and design of overexpression vectors. Each overexpression vector discussed in the paper is listed with its formal name, abbreviated name, and contents. Black boxes represent exons. Intron 1 and exons 1 and 2 were present within the plasmid backbone prior to modification. Checkmarks indicate the presence of artificial intronic flanking sequences. Empty spaces indicate a specific component is not found within that particular vector. TSS: transcription start site.

Figure 3

The Atoh1-HA fusion protein is functional and detectable by immunofluorescence. (A) Detection of Atoh1-HA fusion protein with HA.11 antibody in cells transfected with p183F-Atoh-HA. Scale bar = 100 microns; (B) Illustration of Atoh1 reporter construct; (C) Relative luciferase activity of cells transfected with Atoh1 reporter alone or with the indicated versions of the Atoh1-HA overexpression constructs. Luciferase activities are all referenced to cells transfected only with the reporter construct, which is set at 1.0. All constructs showed a significant increase in luminescence compared to the control except p183F-Atoh1(N162I)-HA. Each bar represents mean (±standard error) within each group. Each experiment was replicated at least three times; (D) Alignment of conserved Atoh1 segment between fly and mouse. Highlighted is the location of the amino acid mutated to make Atoh1 non-functional while maintaining the HA tag. *p < 0.05, **p < 0.005, ***p < 0.0001.

Figure 4

miRNAs are expressed from the 183F-Atoh1-HA vector and functional. (A) p183F-Atoh1-HA transfection leads to production of mature miR-183 family members. Untransfected HEK293T cells and cells transfected with pAtoh1-HA show no detectable expression of 183 family members; whereas miR-183, -96, and -182 are each detected in cells transfected with p183F-Atoh1-HA. U6 levels are provided as the loading control; (B) Illustration of miRNA-specific reporter plasmids. PsiCHECK-2 luciferase reporters contain 2 sites complementary to miR-96, -182, or -183 in the 3′UTR; (C–E) Knockdown of luciferase activity in reporters specific to each member of the miR-183 family; (C) Cells co-transfected with reporter containing miR-183 sites and p183F-Atoh1-HA showed a marked decrease in luciferase activity compared to wells transfected with the miR-183 reporter and pAtoh1-HA. Experiments in (D) and (E) were conducted in the same manner except with miR-96 or miR-182 complementary binding sites in the luciferase reporter. All showed significant decrease in luminescence. Each bar represents mean (±standard error) for each group. Each experiment was replicated at least three times. *p < 0.05, **p < 0.005, ***p < 0.0001.

Figure 5

Plasmid containing miR-183 family and the GFP gene produces functional miRNAs and GFP protein in HEK293T cells. (A) Vector design of p183F-GFP; (B) Visualization of GFP in cells transfected with p183F-GFP. Scale bar = 100 microns; (C) The miR-183 family is expressed from p183F-GFP in mammalian and avian cells. Cells transfected with p183F-GFP showed expression of mature miR-183, -96, and -182. Control (untransfected cells) and pGFP transfected cells exhibit no detectable miRNA. U6 levels serve as the loading control; (D–F) Luciferase activity is decreased by expression of miRNAs from p183F-GFP expressing vector; (D) Cells co-transfected with p183F-GFP and the psiCHECK-2 reporter containing sites complementary to miR-183 show a significant decrease in luciferase activity compared to cells co-transfected with the pGFP and reporter; (E) and (F) show results of experiments similar to (D) except the reporter contained different complementary binding sites: 96 for (E) and 182 for (F). Each bar represents mean (±standard error) for each group. Each experiment was replicated at least three times. *p < 0.05, **p < 0.005, ***p < 0.0001.

Figure 6

The miRNAs produced from both miRNA expression vectors bind to their targets with specificity. (A,B) Luciferase activity is decreased when vectors containing miR-9 are co-expressed with the miR-9 luciferase reporter but not when co-expressed with the miR-183 family expressing plasmids; (A) Luciferase activity of transfected cells containing the miR-9 luciferase reporter with pAtoh1-HA were compared to cells co-transfected with the miR-9 reporter and p183F-Atoh1-HA or p9-Atoh1-HA; (B) Experiments similar to those in A except control cells were co-transfected with miR-9 reporter and pGFP, while experimental cells were co-transfected with reporter and p183F-GFP or p9-GFP. *p < 0.05, **p < 0.005, ***p < 0.0001. Bars represent mean (±standard error) for each group. Each experiment was replicated at least three times.