. 2019 Aug;37(8):884-894.

doi: 10.1038/s41587-019-0205-0. Epub 2019 Aug 2.

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system

Julia F Alterman¹, Bruno M D C Godinho¹, Matthew R Hassler¹, Chantal M Ferguson¹, Dimas Echeverria¹, Ellen Sapp², Reka A Haraszti¹, Andrew H Coles¹, Faith Conroy^{1

3}, Rachael Miller^{1

3}, Loic Roux¹, Paul Yan¹, Emily G Knox¹, Anton A Turanov¹, Robert M King^{4

5}, Gwladys Gernoux⁶, Christian Mueller^{6

7}, Heather L Gray-Edwards⁴, Richard P Moser⁸, Nina C Bishop⁹, Samer M Jaber^{9

10}, Matthew J Gounis⁴, Miguel Sena-Esteves^{6

11}, Athma A Pai¹, Marian DiFiglia², Neil Aronin^{1

3}, Anastasia Khvorova^{12

13}

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.
² Department of Neurology, Massachusetts General Institute for Neurodegenerative Disease, Boston, MA, USA.
³ Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
⁴ Department of Radiology, New England Center for Stroke Research, University of Massachusetts Medical School, Worcester, MA, USA.
⁵ Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA.
⁶ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.
⁷ Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA, USA.
⁸ Department of Neurosurgery, University of Massachusetts Medical School, Worcester, MA, USA.
⁹ Department of Animal Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
¹⁰ Department of Pathology, University of Massachusetts Medical School, Worcester, MA, USA.
¹¹ Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.
¹² RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA. anastasia.khvorova@umassmed.edu.
¹³ Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. anastasia.khvorova@umassmed.edu.

PMID: 31375812
PMCID: PMC6879195
DOI: 10.1038/s41587-019-0205-0

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system

Julia F Alterman et al. Nat Biotechnol. 2019 Aug.

. 2019 Aug;37(8):884-894.

doi: 10.1038/s41587-019-0205-0. Epub 2019 Aug 2.

Authors

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.
² Department of Neurology, Massachusetts General Institute for Neurodegenerative Disease, Boston, MA, USA.
³ Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
⁴ Department of Radiology, New England Center for Stroke Research, University of Massachusetts Medical School, Worcester, MA, USA.
⁵ Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA.
⁶ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA.
⁷ Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA, USA.
⁸ Department of Neurosurgery, University of Massachusetts Medical School, Worcester, MA, USA.
⁹ Department of Animal Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
¹⁰ Department of Pathology, University of Massachusetts Medical School, Worcester, MA, USA.
¹¹ Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA.
¹² RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA. anastasia.khvorova@umassmed.edu.
¹³ Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. anastasia.khvorova@umassmed.edu.

PMID: 31375812
PMCID: PMC6879195
DOI: 10.1038/s41587-019-0205-0

Abstract

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.

PubMed Disclaimer

Conflict of interest statement

COMPETING FINANCIAL INTEREST STATEMENT

AK, JFA, MRH and BMDCG have filed a patent application for branched-oligonucleotides.

Figures

**Figure 1.. A divalent siRNA chemical configuration enables gene silencing in the mouse brain.**
a. Schematic of the chemical structure of Mono-siRNA. b. Biodistribution of Cy3-labeled Mono-siRNA 48 hours after a single intrastriatal injection of 50 μg. Scale bar – 1 mm. c. Schematic of the chemical structure of Di-siRNA. d. Biodistribution of Cy3-labeled Di-siRNA 48 hours after a single intrastriatal injection of 50 μg. Scale bar – 1 mm. e. Image of whole mouse brain injected with Di-siRNA (top – PBS, bottom – Di-siRNA) Scale bar – 0.25 cm. f. Di-siRNA distributes throughout mouse the brain after a bilateral injection of 475 μg (237 μg/ventricle) into the lateral ventricles. Tiled fluorescent images taken 48 hours post injection. Scale bar – 1 mm. **(g,h)** High resolution images of Di-siRNA distribution to various regions of the mouse brain: g. Striatum, h. Cortex. Scale bar – 50 µm. i. Di-siRNA silences huntingtin (HTT) protein at two different doses in multiple brain regions 1 month after bilateral ICV injection. All statistics are One-Way ANOVA with Dunnett’s multiple comparisons test. All results compared to PBS control. Hipp: F(3,17) = 31.92, HTT 60μg ****P<0.0001, HTT 457μg ****P<0.0001. Thal: F(3,17) = 16.875, HTT 60μg ***P=0.0003, HTT 457μg ***P=0.0002. Str: F(3,17) = 33.38, HTT 60μg ****P<0.0001, HTT 457μg ****P<0.0001. PC: F(3,17) = 12.64, HTT 457μg ***P=0.0001. MC: F(3,17) = 53.58, HTT 60μg ***P=0.0003, HTT 475 μg ****P<0.0001. PBS: n=6, NTC n=5, HTT 60 μg: n=5, HTT 475 μg: n=5. Mean ± SD. NTC – non-targeting control. j. Di-siRNA silences apolipoprotein E (APOE) protein 1 month after bilateral ICV injection. All statistics are One-Way ANOVA with Dunnett’s multiple comparisons test. All results compared to PBS control. Hipp: F(2,14) = 1.991, ****P<0.0001. Thal: F(2,14) = 4.283, ****P<0.0001. Str: F(2,15) = 4.781, ****P<0.0001. PC: F(2,14) = 6.618, ****P<0.0001. PBS: n=6. NTC Mean ± SD. NTC – non-targeting control.

**Figure 2.. Di-siRNA efficacy is sustained in mice 6 months after a single bilateral ICV injection.**
Mice were injected with 475 μg siRNA (bilaterally) (237 μg/ventricle) and data was collected at 1, 4, and 6 months post injection. a. Schematic of the duration of effect study in mice. b. Guide strand accumulation (μg/g) in five brain regions. c. Huntingtin (*Htt*) mRNA silencing (% of control) in five brain regions. All statistics are two-tailed unpaired t-tests. All results compared to NTC control. 1 month. Hipp: t=9.56, df=8, ****p<0.0001. Thal: t=10.59, df=8, ****p<0.0001. Str: t=10.63, df=8, ****p<0.0001. PC: t=11.45, df=8, ****p<0.0001. MC: t=8.082, df=8, ****p<0.0001. HTT: n=5, NTC: n=5. 4 month. Hipp: t=14.26, df=9, ****p<0.0001. Thal: t=8.577, df=9, ****p<0.0001. Str: t=6.606, df=9, ****p<0.0001. PC: t=2.561, df=9, *p=0.0306. HTT: n=6, NTC: n=5. 6 month. Hipp: t=6.998, df=7, ***p=0.0002. Thal: t=9.865, df=7, ****p<0.0001. Str: t=4.264, df=7, **p=0.0037. HTT: n=5, NTC: n=4. d. Huntingtin (HTT) protein silencing (% of control) in five brain regions. 1 month. Hipp: t=10.58, df=8, ****p<0.0001. Thal: t=14.092, df=8, **p=0.0035. Str: t=12.05, df=8, ****p<0.0001. PC: t=9.834, df=8, ****p<0.0001. MC: t=10.85, df=8, ****p<0.0001. HTT: n=5, NTC: n=5. 4 month. Hipp: t=6.117, df=9, ***p=0.0002. Thal: t=5.661, df=9, ***p=0.0003. Str: t=6.261, df=9, ***p=0.0001. PC: t=4.651, df=9, **p=0.0012, MC: t=3.46, df=9, **p=0.0072. HTT: n=6, NTC: n=5. 6 month. Hipp: t=19.01, df=7, ****p<0.0001. Thal: t=93.536, df=7, **p=0.0095. Str: t=3.479, df=7, *p=0.0103. HTT: n=5, NTC: n=4. e. Di-siRNA *Htt* mRNA silencing shows a strong correlation with siRNA guide strand accumulation. f. Di-siRNA HTT protein silencing shows a strong correlation with siRNA guide strand accumulation (IC50 ~ 0.5 μg/g). g. siRNA guide strand retention shows strong, two phase tissue clearance kinetics. The majority of the compound is cleared within the first month. Clearance slows between 4 and 6 months. Mean ± SD. NTC – non-targeting control.

**Figure 3.. Gene silencing in the non-human primate CNS with Di-siRNAs.**
Cynomolgus macaques received a unilateral ICV injection of Di-siRNAs (25 mg). Samples were collected at 48 hours for biodistribution and at 30 days for gene silencing and toxicity assessments. a. Schematic of NHP study. b. Image of whole NHP brain (left). c. Images of NHP brain slices (top). Scale bar – 1 cm. High resolution fluorescent images of Di-siRNA in various regions of the NHP brain (bottom) Scale bar – 50 µm. d. Immunofluorescence of the NHP cortex and hippocampus. All images acquired 48 hours after a single unilateral ICV injection of 25 mg Di-siRNA. Hollow arrow indicates glial cells, and closed arrow indicates neurons. Scale bar – 50 µm. e. Quantification of siRNA guide strand (n=4 treated animals, μg/g) in seven brain regions, IL – ipsilateral, CL – contralateral. f. Huntingtin (HTT) protein silencing (% of control) ipsilateral and contralateral sides of four brain regions. Statistics calculated by One-Way ANOVA with Dunnet’s correction for multiple comparisons. All results compared to naïve control. Cortex: F(2,9) = 35.86, ****P<0.0001. Hippocampus: F(2,9) = 35.15, ipsilateral ***P = 0.0001, contralateral ****P<0.0001. Caudate: F(2,9) = 11.51, ipsilateral **P = 0.0028, contralateral **P = 0.0087. Putamen: F(2,9) = 9.08, ipsilateral *P = 0.0385, contralateral **P = 0.0043. n=4/group. 1 month (n=4 Di-siRNA treated animals, n=4 naive animals). All graphs are mean ± SD.

**Figure 4.. Di-siRNA enables silencing in the spinal cord of non-human primates.**
a. Image of the whole NHP spinal cord. b. Tiled fluorescent images of cross sections of the spinal cord at each segment. Scale bar – 1 mm. c. Immunofluorescence of the NHP spinal cord (cervical, thoracic and lumbar regions). All images acquired 48 hours after a single unilateral ICV injection of 25 mg Di-siRNA. Scale bar – 50 µm. d. Quantification of siRNA guide strand (μg/g) in three spinal cord regions, both white and grey matter. e. HTT mRNA silencing in various regions of the NHP spinal cord. Statistics calculated by two-tailed unpaired t-test: Cervical GM: t=4.686, df=6, **P=0.0034. Thoracic WM: t= 5.278, df=6, **P=0.0019. Thoracic GM: t=5.757, df=6, **P=0.0012. Lumbar WM: t=4.69, df=6, **P=0.0034. Lumbar GM: t=9.853, df=6, ****P<0.0001. 1 month (n=4 Di-siRNA treated animals, n=4 naive animals). All graphs are mean ± SD.

**Figure 5.. Assessment of brain toxicity 1 month after a single ICV injection of Di-siRNA in non-human primates.**
Animals were all dosed with 25 mg Di-siRNA unilaterally into the lateral ventricle. a. Pre-operative and post-operative (30 days) MRI scans. b. *GFAP mRNA* expression was assessed 30 days after a single ICV injection. c. *IBA-1* mRNA expression 30 days after a single ICV injection. Statistics calculated by One-Way ANOVA with Dunnet’s correction for multiple comparisons. All results compared to naïve control. F(2,9) = 4.578 iplsilateral *P<0.0359. n=4/group.

**Figure 6.. RNA-seq shows limited off-target effects of Di-siRNA.**
RNA collected from cynomolgus macaques was subjected to RNA-sequencing to assess genome-wide patterns of differential gene expression. a. Volcano plot showing genome-wide gene expression changes in Di-siRNA^HTT-treated NHPs, with differentially-expressed genes (Benjamini Hochberg FDR < 1%) in orange and differentially-expressed genes with a 3’ UTR seed complementary region in red. Enrichment of 3’ UTR seed complementarity down- or up-regulated genes (top-right) was calculated using a Fisher’s exact test. b. Volcano plot showing unbiased screen for enriched 6-mer sequences within 3’ UTRs of differentially-expressed gene (FDR < 1%). There are no significantly over- or under-represented sequences after Benjamini Hochberg multiple test correction. Seed complementary sequence for Di-siRNA^HTT is indicated in black. c. Gene expression measurements (transcripts per million, x-axis) across 4 naïve NHPs (grey) and 4 Di-siRNA^HTT treated NHPs (red) for the 8 differentially expressed genes with seed complementary regions. d. Significant gene ontology categories for differentially expressed genes with FDR < 1% and FDR < 5%.

See this image and copyright information in PMC

References

1. Khvorova A & Watts JK The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol 35, 238–248 (2017). - PMC - PubMed
1. Ostergaard ME et al. Efficient Synthesis and Biological Evaluation of 5’-GalNAc Conjugated Antisense Oligonucleotides. Bioconjug Chem 26, 1451–1455 (2015). - PubMed
1. Rajeev KG et al. Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo. Chembiochem : a European journal of chemical biology 16, 903–908 (2015). - PubMed
1. Kordasiewicz HB et al. Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis. Neuron 74, 1031–1044 (2012). - PMC - PubMed
1. Finkel RS et al. Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy. N Engl J Med 377, 1723–1732 (2017). - PubMed

Publication types

Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- The Lens - Patent Citations Database
Molecular Biology Databases
- NIAID Data Ecosystem - Find datasets on Infectious and Immune-mediated Diseases

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system

Affiliations

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases