Therapeutic reversal of Huntington's disease by in vivo self-assembled siRNAs

Li Zhang, Tengteng Wu, Yangyang Shan, Ge Li, Xue Ni, Xiaorui Chen, Xiuting Hu, Lishan Lin, Yongchao Li, Yalun Guan, Jinfeng Gao, Dingbang Chen, Yu Zhang, Zhong Pei, Xi Chen, Li Zhang, Tengteng Wu, Yangyang Shan, Ge Li, Xue Ni, Xiaorui Chen, Xiuting Hu, Lishan Lin, Yongchao Li, Yalun Guan, Jinfeng Gao, Dingbang Chen, Yu Zhang, Zhong Pei, Xi Chen

Abstract

Huntington's disease is an autosomal-dominant neurodegenerative disease caused by CAG expansion in exon 1 of the huntingtin (HTT) gene. Since mutant huntingtin (mHTT) protein is the root cause of Huntington's disease, oligonucleotide-based therapeutic approaches using small interfering RNAs (siRNAs) and antisense oligonucleotides designed to specifically silence mHTT may be novel therapeutic strategies for Huntington's disease. Unfortunately, the lack of an effective in vivo delivery system remains a major obstacle to realizing the full potential of oligonucleotide therapeutics, especially regarding the delivery of oligonucleotides to the cortex and striatum, the most severely affected brain regions in Huntington's disease. In this study, we present a synthetic biology strategy that integrates the naturally existing exosome-circulating system with artificial genetic circuits for self-assembly and delivery of mHTT-silencing siRNA to the cortex and striatum. We designed a cytomegalovirus promoter-directed genetic circuit encoding both a neuron-targeting rabies virus glycoprotein tag and an mHTT siRNA. After being taken up by mouse livers after intravenous injection, this circuit was able to reprogramme hepatocytes to transcribe and self-assemble mHTT siRNA into rabies virus glycoprotein-tagged exosomes. The mHTT siRNA was further delivered through the exosome-circulating system and guided by a rabies virus glycoprotein tag to the cortex and striatum. Consequently, in three mouse models of Huntington's disease treated with this circuit, the levels of mHTT protein and toxic aggregates were successfully reduced in the cortex and striatum, therefore ameliorating behavioural deficits and striatal and cortical neuropathologies. Overall, our findings establish a convenient, effective and safe strategy for self-assembly of siRNAs in vivo that may provide a significant therapeutic benefit for Huntington's disease.

Keywords: Huntington’s disease; exosome; self-assembly; siRNA; synthetic biology.

© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.

Figures

Figure 1
Figure 1
Schematic description of the architecture of the genetic circuit. The genetic circuit contains three different functional modules: a co-driving CMV promoter, a neuron-guiding RVG tag and an mHTT siRNA-expressing backbone. When the genetic circuit is placed in a tissue chassis such as the liver, the CMV promoter drives the transcription of mHTT siRNA and facilitates loading of the payload of mHTT siRNA into exosomes as cargo; simultaneously, the CMV promoter directs the localization of the RVG guidance tag onto the exosome surface to confer neuron-targeting properties on the exosomes. After being released into the circulation and delivered through the exosome-circulating system, the mHTT siRNA enclosed in RVG-tagged exosomes is guided by the RVG tag to penetrate the blood–brain barrier (BBB) and arrive at the cortex and striatum, ultimately resulting in mRNA degradation and decreased protein expression of the mHTT gene.
Figure 2
Figure 2
Characterization of self-assembled mHTT siRNA in an ex vivo model. (A) Schematic of the experimental design. C57BL/6J mice were intravenously injected with PBS or with 5 mg/kg CMV-scrR, CMV-siRmHTT or CMV-RVG-siRmHTT circuit every 2 days for a total of seven times, and then the exosomes were purified from mouse plasma and incubated with HEK293T cells. Next, uptake of self-assembled mHTT siRNA by HEK293T cells and the subsequent suppression of mHTT expression and aggregation by self-assembled mHTT siRNA were examined in this ex vivo model. (B) Quantitative RT-PCR analysis of mHTT siRNA levels in purified exosomes (n = 5 in each group). (C) Purified exosomes were fluorescently labelled with PKH26, and PKH26-labelled exosomes were incubated with HEK293T cells for 6 h. The levels of intracellular fluorescence intensity were monitored by confocal microscopy. (D) Quantitation of the fluorescence intensity shown in C (n = 5 in each group). (E) Western blot analysis of mHTT-FLAG protein levels in mHTT-Q66-expressing HEK293T cells after incubation with purified exosomes. HEK293T cells were stably transfected with LV-mHTT-Q66-FLAG ahead of incubation. (F) Quantitation of the mHTT-FLAG protein levels shown in E (n = 5 in each group). (G) Quantitative RT-PCR analysis of mHTT mRNA levels in mHTT-Q66-expressing HEK293T cells after incubation with purified exosomes (n = 5 in each group). (H) HEK293T cells coexpressing mHTT-Q66 and GFP were incubated with purified exosomes. Fluorescence microscopy was used to assess mHTT aggregates (bright green puncta). Scale bar = 5 μm. (I) Quantitation of the aggregated mHTT dots shown in H (n = 5 in each group). (J) Purified exosomes were fluorescently labelled with PKH26, and PKH26-labelled exosomes were incubated with STHdh-Q111 cells for 6 h. The levels of intracellular fluorescence intensity were monitored by confocal microscopy. Scale bar = 20 μm. (K) Western blot analysis of mHTT protein levels in STHdh-Q111 cells after incubation with purified exosomes. STHdh-Q7 cells expressing wild-type HTT served as a control. (L) Quantitation of the HTT protein levels shown in K (n = 5 in each group). Values are presented as mean ± SEM. Significance was determined using one-way ANOVA followed by Bonferroni's multiple comparisons in F, G, I and L. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns = not significant.
Figure 3
Figure 3
Tracking and visualization of the delivery of self-assembled mHTT siRNA into the cortex and striatum. (A–C) Quantitative RT–PCR analysis of mHTT siRNA levels in the liver, plasma and cortex/striatum after intravenous injection of C57BL/6J mice with PBS or with 5 mg/kg CMV-scrR, CMV-siRmHTT or CMV-RVG-siRmHTT circuit for a total of three times (n = 5 in each group). Values are presented as the means ± SEM. (D and E) In situ detection of mHTT siRNA in the mouse cortex and striatum. C57BL/6J mice were intravenously injected with PBS or with 5 mg/kg CMV-scrR, CMV-siRmHTT or CMV-RVG-siRmHTT circuit for a total of three times. Positive in situ hybridization signals in the cortex and striatum are shown in green, and DAPI-stained nuclei are shown in blue. Scale bar = 50 μm. (F and G) Direct visualization of the suppression of GFP fluorescence levels in vivo by self-assembled GFP siRNA. GFP-transgenic mice were intravenously injected with PBS or with 5 mg/kg CMV-scrR, CMV-siRGFP or CMV-RVG-siRGFP circuit every 2 days for a total of seven times. After treatment termination, mice were killed and GFP fluorescence levels were assessed in frozen sections of the cortex and striatum. Representative fluorescence microscopy images are shown. Positive GFP signals are shown in green, NeuN-stained neurons are shown in red and DAPI-stained nuclei are shown in blue. Scale bar = 100 μm.
Figure 4
Figure 4
Evaluation of the therapeutic efficacy of self-assembled mHTT siRNA in N171-82Q and BACHD Huntington’s disease models. (A) Schematic of the experimental design. At 8 weeks of age, N171-82Q mice were intravenously injected with 5 mg/kg CMV-scrR or CMV-RVG-siRmHTT circuit every 2 days for a total of 2 weeks. After treatment termination, the mice were monitored to evaluate behavioural performance and mHTT accumulation. (B) Motor performance in a rotarod test. Non-transgenic littermates were included as behavioural controls (n = 8 in each group). (C) Western blot analysis of mHTT protein levels in the cortex and striatum. α-Tubulin served as the internal loading control. (D) Quantitation of the mHTT protein levels shown in C (n = 4 in cortex; n = 8 in striatum). (E) Quantitative RT-PCR analysis of mHTT mRNA levels in the cortex and striatum (n = 7–8 in each group). (F) Schematic of the experimental design. At 3 months of age, BACHD mice were intravenously injected with 5 mg/kg CMV-scrR or CMV-RVG-siRmHTT circuit every 2 days for a total of 2 weeks. After treatment termination, the mice were monitored to evaluate mHTT accumulation and neuropathology. (G) Western blot analysis of mHTT protein levels in the cortex and striatum. The more slowly migrating band is the human mHTT protein, and the more rapidly migrating band is mouse endogenous HTT. Endogenous HTT and α-tubulin served as the internal loading control. (H) Quantitation of the mHTT protein levels shown in G (n = 8 in each group). (I) Quantitative RT–PCR analysis of mHTT mRNA levels in the cortex and striatum (n = 8 in each group). (J) Immunofluorescence staining of aggregated mHTT (EM48, green), neurons (NeuN, red) and nuclei (DAPI, blue) in striatal and cortical sections. Scale bar = 50 μm. Values are presented as the means ± SEM. Significance was determined using two-sided t-test in D, E, H and I, or using one-way ANOVA followed by Bonferroni's multiple comparisons in B. *P < 0.05; ***P < 0.001; ****P < 0.0001; ns = not significant.
Figure 5
Figure 5
Evaluation of the long-term therapeutic efficacy of self-assembled mHTT siRNA in the YAC128 Huntington’s disease model. (A) Schematic of the experimental design. At 6 weeks of age, YAC128 mice were intravenously injected with 5 mg/kg CMV-scrR or CMV-RVG-siRmHTT circuit twice a week for a total of 8 weeks. After treatment termination, the mice were monitored to evaluate behavioural performance, mHTT accumulation and neuropathology. (B) Motor performance on a rotarod test. Non-transgenic littermates were included as behavioural controls (n = 8 in each group). (C) Western blot analysis of mHTT protein levels in the cortex and striatum. The more slowly migrating band is the human mHTT protein, and the more rapidly migrating band is mouse endogenous HTT. Endogenous HTT served as the internal loading control. (D) Quantitation of the mHTT protein levels shown in (C) (n = 8 in each group). (E) Quantitative RT–PCR analysis of mHTT mRNA levels in the cortex and striatum (n = 8 in each group). (F) Immunofluorescence staining of aggregated mHTT (EM48, green), neurons (NeuN, red) and nuclei (DAPI, blue) in striatal and cortical sections. Scale bar = 50 μm. Values are presented as mean ± SEM. Significance was determined using two-sided t-test in D and E, or using one-way ANOVA followed by Bonferroni's multiple comparisons in B. ***P < 0.001; ****P < 0.0001; ns = not significant.

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