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. 2012 May;14(5):299-315.
doi: 10.1002/jgm.2626.

Lentiviral Vectors Encoding Short Hairpin RNAs Efficiently Transduce and Knockdown LINGO-1 but Induce an Interferon Response and Cytotoxicity in Central Nervous System Neurones

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Free PMC article

Lentiviral Vectors Encoding Short Hairpin RNAs Efficiently Transduce and Knockdown LINGO-1 but Induce an Interferon Response and Cytotoxicity in Central Nervous System Neurones

Thomas H Hutson et al. J Gene Med. .
Free PMC article

Abstract

Background: Knocking down neuronal LINGO-1 using short hairpin RNAs (shRNAs) might enhance axon regeneration in the central nervous system (CNS). Integration-deficient lentiviral vectors have great potential as a therapeutic delivery system for CNS injuries. However, recent studies have revealed that shRNAs can induce an interferon response resulting in off-target effects and cytotoxicity.

Methods: CNS neurones were transduced with integration-deficient lentiviral vectors in vitro. The transcriptional effect of shRNA expression was analysed using quantitative real time-polymerase chain reaction and northern blots were used to assess shRNA production.

Results: Integration-deficient lentiviral vectors efficiently transduced CNS neurones and knocked down LINGO-1 mRNA in vitro. However, an increase in cell death was observed when lentiviral vectors encoding an shRNA were applied or when high vector concentrations were used. We demonstrate that high doses of vector or the use of vectors encoding shRNAs can induce an up-regulation of interferon-stimulated genes (2',5'-oligoadenylate synthase 1 and protein kinase R although not myxovirus resistance 1) and a down-regulation of off-target genes (including p75(NTR) and Nogo receptor 1). Furthermore, the northern blot demonstrated that these negative consequences occur even when lentiviral vectors express low levels of shRNAs. Taken together, these results may explain why neurite outgrowth was not enhanced on an inhibitory substrate following transduction with lentiviral vectors encoding an shRNA targeting LINGO-1.

Conclusions: These findings highlight the importance of including appropriate controls to verify silencing specificity and the requirement to check for an interferon response when conducting RNA interference experiments. However, the potential benefits that RNA interference and viral vectors offer to gene-based therapies to CNS injuries cannot be overlooked and demand further investigation.

Conflict of interest statement

Conflict of Interest Statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the integration-deficient lentiviral vectors. Schematic depicts linear form of double stranded DNA after completion of reverse transcription. All vectors contain an eGFP expression cassette under the control of a CMV promoter and including a WPRE. The cassettes are flanked by the self-inactivating 5’ deleted LTR. (A) Lenti-LINGO1-sh1-4/Lenti-Scr contain an shRNA expression cassette driven by the H1 promoter placed downstream of the eGFP expression cassette. (B) Lenti-H1 contains the H1 promoter but no shRNA or Pol III termination sequence. dLTR, deleted long terminal repeat; GLS, gag leader sequence incorporating the packaging element (y); RRE, rev response element; cPPT/cTS, central polypurine tract and central termination sequence; CMV, cytomegalovirus immediate early promoter; eGFP, enhancer green fluorescent protein; H1, H1-RNA promoter; shRNA, short hairpin RNA; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element.
Figure 2
Figure 2
CGNs are efficiently transduced in vitro, although viability was affected by shRNAs and viral concentration. (A) Quantification of the mean number of transduced CGNs using Lenti-LINGO1-sh4. The number of eGFP positive CGNs was counted and the mean number of transduced CGNs plotted for each MOI. There were significantly more CGNs transduced using an MOI 10 compared to MOI 1 or 50 (** P < 0.01). Values represent mean and SEM; analysis was performed using one way ANOVA with Tukey post-hoc tests, n = 3. (B) Quantification of the percentage transduction efficiency, which was calculated by dividing the number of transduced CGNs by the total number CGNs multiplied by one hundred. Percentage transduction did not change with viral concentrations higher than MOI 10 where the transduction efficiency was already above 90%. Values represent mean and SEM, n = 3. (C) Quantification of the mean number of CGNs after transduction with Lenti-LINGO1-sh4 or Lenti-H1. The number of CGNs was counted and the mean number plotted for each MOI. Neuronal viability was affected by MOI and there was a decrease in neuronal viability at higher viral concentrations. Neuronal viability was also differentially affected by the viral vectors. There were different patterns of neuronal viability between the lentiviral vectors that encoded shRNAs and the viral vectors that did not encode an shRNA. Values represent mean and SEM, analysis was performed using one way ANOVA with Tukey post-hoc tests, n = 3/group. (D) CGNs are efficiently transduced by Lenti LINGO1-sh4 at an MOI of 10. Transduced CGNs expressing eGFP (green) and stained for beta-III tubulin (red) appear yellow. Scale bar: 100 μm.
Figure 3
Figure 3
Lenti-LINGO1-sh4 knocks down LINGO-1 mRNA, although non-specific silencing was also observed with the control vectors. (A) Quantification of the relative level of LINGO-1 mRNA in CGNs transduced with lentiviral vectors at an MOI 50. Lenti-LINGO1-sh4 significantly knocked down LINGO-1 expression 2.1 fold and 2.2 fold respectively compared to Lenti-Scr and Lenti-H1 (* P < 0.05). In addition, significantly lower levels of LINGO-1 were detected in CGNs transduced with any of the lentiviral vectors compared to the NVC (*** P < 0.001). Values represent mean and SEM, analysis was performed using one way ANOVA with Tukey post-hoc tests, n = 4/group. (B) Quantification of the relative level of LINGO-1 mRNA in CGNs after transduction at an MOI 10. Lenti-LINGO1-sh4 significantly knocked down LINGO-1 expression 2.2 fold and 2.3 fold respectively compared to Lenti-Scr, Lenti-H1 (* P < 0.05). However, significantly lower levels of LINGO-1 were detected in CGNs transduced with any of the lentiviral vectors compared to the NVC (*** P < 0.001). Values represent mean and SEM, analysis was performed using one way ANOVA with Tukey post-hoc tests, n = 3/group.
Figure 4
Figure 4
Integration-deficient lentiviral vectors encoding an shRNA targeting LINGO-1 do not enhance neurite outgrowth. (A) The neurite length of beta-III tubulin positive CGNs was measured, divided by the total number of neurons and the mean neurite length per neuron plotted. CGNs plated on the CHO-R2 cells had significantly longer neurites compared to CGNs plated on the inhibitory CHO-MAG cells (*** P < 0.001). CHO-MAG inhibition could be partially reversed with using 50 μM Y 27632 (*** P < 0.001). Lenti-LINGO1-sh4 did not significantly increase neurite outgrowth of CGNs plated on an inhibitory MAG substrate compared to the control lentiviral vectors or the NVC (P > 0.05). Values represent mean and SEM, analysis was performed using one way ANOVA, with Dunnett’s post-hoc tests comparing to CHO-MAG cells, P < 0.001, n = 8/group. (B) CGN neurite outgrowth is inhibited by CHO-MAG cells. (C) Transduction with Lenti-LINGO1-sh4 does not alleviate the MAG inhibition and enhance CGN neurite outgrowth. (D) The ROCK inhibitor Y-27632 partially reverses the MAG inhibition and promotes neurite outgrowth. Scale bar: 100 μm.
Figure 5
Figure 5
OAS1 and PKR induction following transduction with integration-deficient lentiviral vectors encoding shRNAs. (A) Quantification of the relative level of OAS1 mRNA in CGNs transduced using an MOI 10. There was a significant increase in OAS1 expression with Lenti-LINGO1-sh1, Lenti-LINGO1-sh2 and Lenti-LINGO1-sh4 compared to the NVC. Values represent mean and SEM, analysis was performed using one way ANOVA with Tukey post-hoc tests, * P < 0.05, ** P < 0.01, n = 3/group. (B) Quantification of the relative level of OAS1 mRNA in CGNs transduced using an MOI 50. All the lentiviral vectors significantly increased OAS1 expression compared to the NVC. Values represent mean and SEM, analysis was performed using Kruskal-Wallis with Mann-Whitney post-hoc tests, * P < 0.05, n = 4/group. (C) Quantification of the relative level of PKR mRNA in CGNs transduced using an MOI 10. PKR expression was not significantly affected compared to the NVC. Values represent mean and SEM, analysis was performed using one way ANOVA, P > 0.05, n = 3/group. (D) Quantification of the relative level of PKR mRNA in CGNs transduced using an MOI 50. PKR expression was significantly increased in CGNs transduced with any of the shRNA expressing lentiviral vectors compared to the NVC. Values represent mean and SEM, analysis was performed using Kruskal-Wallis with Mann Whitney post-hoc tests, * P < 0.05, n = 4/group. (E) Quantification of the relative level of Mx1 mRNA in CGNs transduced at an MOI 10. No significant difference in Mx1 expression was observed compared to the NVC. Values represent mean and SEM, analysis was performed using one way ANOVA, P > 0.05, n = 3/group. (F) Quantification of the relative level of Mx1 mRNA in CGNs transduced using an MOI 50. Mx1 expression was not significantly affected compared to the NVC. Values represent mean and SEM, analysis was performed using Kruskal-Wallis, P > 0.05, n = 4/group.
Figure 6
Figure 6
Off-target genes p75NTR and NgR1 are reduced following lentiviral vector transduction. (A) Quantification of the relative level of p75NTR mRNA in CGNs transduced using an MOI 10. There was a significant decrease in p75NTR expression with Lenti-LINGO1-sh2, Lenti-LINGO1-sh3 and Lenti-Scr compared to the NVC. Values represent mean and SEM, analysis was performed using one way ANOVA with Dunnett’s post-hoc tests comparing to the NVC, * P < 0.05, n = 4/group. (B) Quantification of the relative level of p75NTR mRNA in CGNs transduced using an MOI 50. p75NTR expression was significantly reduced in CGNs transduced with Lenti-LINGO1-sh2, Lenti-LINGO1-sh3, Lenti-LINGO1-sh4 and Lenti-H1 compared to the NVC. Values represent mean and SEM, analysis was performed using one way ANOVA with Dunnett’s post-hoc tests comparing to the NVC, * P < 0.05, ** P < 0.01 n = 4/group. (C) Quantification of the relative level of NgR1 mRNA in CGNs transduced using an MOI 10. NgR1 expression was significantly decreased in CGNs transduced with Lenti-LINGO1-sh2, Lenti-LINGO1-sh3, Lenti-LINGO1-sh4 and Lenti-Scr compared to the NVC. Values represent mean and SEM, analysis was performed using one way ANOVA with Dunnett’s post-hoc tests comparing to the NVC, * P < 0.05, n = 4/group (D) Quantification of the relative level of NgR1 mRNA in CGNs transduced using an MOI 50. There was a significant decrease in NgR1 expression with any of the lentiviral vectors compared to the NVC. Values represent mean and SEM, analysis was performed using one way ANOVA with Dunnett’s post-hoc tests comparing to the NVC, * P < 0.05, ** P < 0.01, *** P < 0.001, n = 4/group.
Figure 7
Figure 7
Northern blots do not detect a high level of shRNA expression. (A) Image of eGFP positive HEK cells that were efficiently transduced using Lenti-LINGO1-sh4 at an MOI 10. Scale bar: 500 μm. (B) Image of eGFP positive HEK cells that were efficiently transfected with the LINGO1-sh4 plasmid using PEI. (C) Image of the resolved RNA gel, the bands correspond to the different ribosomal RNA (rRNA) species or transfer RNAs (tRNAs) as labelled on the left side. The RNA resolved from the plasmid transfected HEK cells shows some smearing. The shRNA (white arrowhead) and siRNA (black arrowhead) positive controls can be seen, confirming the design and sensitivity of the probe (D) Image of the small transcript northern blot. Neither the shRNA nor siRNA was detected from the virus transduced HEK cell RNA. A faint band representing the shRNA was detected from the plasmid transfected HEK cell RNA but a band for the siRNA was not detected. Bands representing the shRNA (white arrowhead) and siRNA (black arrowhead) positive controls were detected.

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