Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier

doi:10.1016/j.ymthe.2018.08.005

. 2018 Nov 7;26(11):2580-2591.

doi: 10.1016/j.ymthe.2018.08.005. Epub 2018 Aug 8.

Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: bruno.godinho@umassmed.edu.
² Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01605, USA.
³ Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁴ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁵ Department of Neurology, Mass General Institute for Neurodegenerative Disease, Charlestown, MA 02129, USA.
⁶ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁷ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: anastasia.khvorova@umassmed.edu.

PMID: 30143435
PMCID: PMC6225091
DOI: 10.1016/j.ymthe.2018.08.005

Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier

Bruno M D C Godinho et al. Mol Ther. 2018.

. 2018 Nov 7;26(11):2580-2591.

doi: 10.1016/j.ymthe.2018.08.005. Epub 2018 Aug 8.

Authors

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: bruno.godinho@umassmed.edu.
² Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01605, USA.
³ Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁴ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁵ Department of Neurology, Mass General Institute for Neurodegenerative Disease, Charlestown, MA 02129, USA.
⁶ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁷ RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: anastasia.khvorova@umassmed.edu.

PMID: 30143435
PMCID: PMC6225091
DOI: 10.1016/j.ymthe.2018.08.005

Abstract

Effective transvascular delivery of therapeutic oligonucleotides to the brain presents a major hurdle to the development of gene silencing technologies for treatment of genetically defined neurological disorders. Distribution to the brain after systemic administrations is hampered by the low permeability of the blood-brain barrier (BBB) and the rapid clearance kinetics of these drugs from the blood. Here we show that transient osmotic disruption of the BBB enables transvascular delivery of hydrophobically modified small interfering RNA (hsiRNA) to the rat brain. Intracarotid administration of 25% mannitol and hsiRNA conjugated to phosphocholine-docosahexanoic acid (PC-DHA) resulted in broad ipsilateral distribution of PC-DHA-hsiRNAs in the brain. PC-DHA conjugation enables hsiRNA retention in the parenchyma proximal to the brain vasculature and enabled active internalization by neurons and astrocytes. Moreover, transvascular delivery of PC-DHA-hsiRNAs effected Htt mRNA silencing in the striatum (55%), hippocampus (51%), somatosensory cortex (52%), motor cortex (37%), and thalamus (33%) 1 week after administration. Aside from mild gliosis induced by osmotic disruption of the BBB, transvascular delivery of PC-DHA-hsiRNAs was not associated with neurotoxicity. Together, these findings provide proof-of-concept that temporary disruption of the BBB is an effective strategy for the delivery of therapeutic oligonucleotides to the brain.

Keywords: RNA interference; hyperosmolar mannitol; intracarotid injection; osmotic disruption; therapeutic oligonucleotide.

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Figures

**Figure 1**
Blood-Brain Barrier Disruption Approach for Transvascular Delivery of PC-DHA-hsiRNAs to the Rat Brain Schematic illustrating cannulation and intracarotid administration of mannitol and PC-DHA-hsiRNAs to the rat brain. Anesthetized rats were placed in a supine position and the carotid artery exposed at the level of the neck. The external carotid artery (ECA) was distally ligated and the cannula advanced to the bifurcation of the common carotid artery (CCA). The ECA stump was rotated ∼180° to enable alignment with the proximal end of the internal carotid artery (ICA). The proximal end of the CCA was transiently clamped during injections. Mannitol (25%) was administered to disrupt the blood-brain barrier (BBB), followed by phosphocholine-docosahexanoic acid (PC-DHA) hydrophobic siRNAs (hsiRNA) 5 min after. (box, top) Chemical structure of PC-DHA. (box, bottom) PyMOL molecular model of PC-DHA-hsiRNAs. PC-DHA is conjugated to the 3′ end of the sense strand. The asymmetric siRNA is fully chemically modified with alternating 2′-O-methyl and 2′-fluoro sugar modifications and phosphorothioate backbone linkages. The antisense strand contained 5′-phosphate modification. After all injections, the proximal end of the ECA was ligated and catheters removed.

**Figure 2**
PC-DHA-hsiRNAs Show Widespread Distribution in the Rat Brain following Mannitol-Induced Disruption of the Blood-Brain Barrier Cy3-labeled PC-DHA-hsiRNAs (37 mg/kg) were administered through the right carotid artery preceded by saline or mannitol, and rats were euthanized 48 hr after injections. (A) Tiled fluorescent images (20× objective) were obtained from coronal sections. The schematic at the top shows the approximate positions of the anterior (red line) and posterior (blue line) sections. Cyan: nuclei (DAPI); red: Cy3-labeled PC-DHA-hsiRNA. Scale bar, 2 mm. (B) Concentrations of PC-DHA-hsiRNA guide strands were quantified by PNA-based hybridization assay. Tissues were assayed from injected (top) and non-injected (bottom) sides of the brain. n = 2–3 animals/group, mean ± SD. CER, cerebellum; HIP, hippocampus; MC, motor cortex; PFC, pre-frontal cortex; SC, somatosensory cortex; ST, striatum; THAL, thalamus.

**Figure 3**
PC-DHA-hsiRNAs Localize to Neurons and Glia after Transvascular Delivery Cy3-labeled PC-DHA-hsiRNAs (37 mg/kg) were administered through the right carotid artery preceded by saline or mannitol, and rats were euthanized 48 hr after injections. High-magnification (63× objective) fluorescent images were acquired from representative regions of the brain, including motor cortex (cortex), caudate putamen (striatum), thalamic nucleus (thalamus), and dentate gyrus (hippocampus). Nuclei (DAPI, cyan), astrocytes (GFAP, magenta), neurons (NeuN, green), and hsiRNAs (Cy3, red) were labeled. Filled arrowheads indicate binding to the neurovascular unit of the BBB. Unfilled arrowheads indicate perinuclear localization of PC-DHA-hsiRNAs within neurons (white arrowheads) and astrocytes (yellow arrowheads). Scale bars, 40 μm.

**Figure 4**
PC-DHA-hsiRNAs Enable Gene Silencing in Various Brain Regions after Transvascular Delivery PC-DHA-hsiRNAs (16 mg/kg) or saline was administered through the right carotid artery after pre-treatment with mannitol. Huntingtin (HTT)-targeting (PC-DHA-hsiRNA^HTT) and non-targeting control (PC-DHA-hsiRNA^NTC) sequences were used. Gene expression was assessed from tissue punch biopsies from the injected side 7 days post-injection. Data were normalized to a housekeeping gene (cyclophilin β) and presented as a percentage of saline control. n = 7–8 animals/group, mean ± SD. One-way ANOVA with Tukey’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, PC-DHA-hsiRNA^HTT versus saline control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, PC-DHA-hsiRNA^HTT versus PC-DHA-hsiRNA^NTC.

**Figure 5**
Transvascular Delivery of PC-DHA-hsiRNA Reduces HTT Protein in the Rat Brain PC-DHA-hsiRNAs (16 mg/kg) or saline was administered through the right carotid artery after pre-treatment with mannitol. Huntingtin (HTT)-targeting (PC-DHA-hsiRNA^HTT) and non-targeting control (PC-DHA-hsiRNA^NTC) sequences were used. Levels of HTT and glial fibrillary acidic protein (GFAP) expression were assessed by western blots 7 days post-injection. (A) Representative blots depicting protein levels from tissue biopsies collected from the ipsilateral thalamus. (B) Densitometry analysis of HTT protein levels normalized to β-actin and expressed relatively to saline control. n = 7–8 animals/group, mean ± SD. One-way ANOVA with Tukey’s post hoc test: *p < 0.05, PC-DHA-hsiRNA^HTT versus saline; ^#p < 0.05, PC-DHA-hsiRNA^HTT versus PC-DHA-hsiRNA^NTC.

**Figure 6**
Delivery of PC-DHA-hsiRNA through the External Carotid Artery Limits Apoptosis in the Rat Striatum Cy3-labeled PC-DHA-hsiRNAs were administered through the right carotid artery preceded by saline or mannitol, and rats were euthanized 48 hr after injections. Schematic to the right illustrates the direct administration (15 mg/kg) carried out through the common carotid artery (CCA) and the indirect injection (37 mg/kg) performed by cannulation of the external carotid artery (ECA). The respective arteries were permanently ligated after administrations at the sites indicated by the blue arrow (CCA) and green arrow (ECA). Fluorescent images (20× objective) of the striatum show PC-DHA-hsiRNA (cy3, red), nuclei (DAPI, cyan), and apoptotic nuclei (TUNEL, green). The DNase-treated sample was used as TUNEL-positive control. n = 2–3 animals/group. Scale bars, 100 μm.

**Figure 7**
Delivery of PC-DHA-hsiRNA through the External Carotid Artery Limits Apoptosis in the Rat Thalamus Cy3-labeled PC-DHA-hsiRNAs were administered through the right carotid artery preceded by saline or mannitol, and rats were euthanized 48 hr after injections. Schematic to the right illustrates the direct administration (15 mg/kg) carried out through the common carotid artery (CCA) and the indirect injection (37 mg/kg) performed by cannulation of the external carotid artery (ECA). The respective arteries were permanently ligated after administrations at the sites indicated by the blue arrow (CCA) and green arrow (ECA). Fluorescent images (20× objective) of the thalamus show PC-DHA-hsiRNA (cy3, red), nuclei (DAPI, cyan), and apoptotic nuclei (TUNEL, green). The DNase-treated sample was used as TUNEL-positive control. n = 2–3 animals/group. Scale bars, 100 μm.

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Comment in

Gene Silencing in the Right Place at the Right Time.
Gutkin A, Peer D. Gutkin A, et al. Mol Ther. 2018 Nov 7;26(11):2539-2541. doi: 10.1016/j.ymthe.2018.10.005. Epub 2018 Oct 23. Mol Ther. 2018. PMID: 30366821 Free PMC article. No abstract available.

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References

1. Haraszti R.A., Roux L., Coles A.H., Turanov A.A., Alterman J.F., Echeverria D., Godinho B.M.D.C., Aronin N., Khvorova A. 5′-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo. Nucleic Acids Res. 2017;45:7581–7592. - PMC - PubMed
1. Hassler M.R., Turanov A.A., Alterman J.F., Haraszti R.A., Coles A.H., Osborn M.F., Echeverria D., Nikan M., Salomon W.E., Roux L. Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo. Nucleic Acids Res. 2018;46:2185–2196. - PMC - PubMed
1. Pasi K.J., Rangarajan S., Georgiev P., Mant T., Creagh M.D., Lissitchkov T., Bevan D., Austin S., Hay C.R., Hegemann I. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N. Engl. J. Med. 2017;377:819–828. - PubMed
1. Ray K.K., Landmesser U., Leiter L.A., Kallend D., Dufour R., Karakas M., Hall T., Troquay R.P., Turner T., Visseren F.L. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 2017;376:1430–1440. - PubMed
1. Alterman J.F., Hall L.M., Coles A.H., Hassler M.R., Didiot M.-C., Chase K., Abraham J., Sottosanti E., Johnson E., Sapp E. Hydrophobically modified siRNAs silence Huntingtin mRNA in primary neurons and mouse brain. Mol. Ther. Nucleic Acids. 2015;4:e266. - PMC - PubMed

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[1] Haraszti R.A., Roux L., Coles A.H., Turanov A.A., Alterman J.F., Echeverria D., Godinho B.M.D.C., Aronin N., Khvorova A. 5′-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo. Nucleic Acids Res. 2017;45:7581–7592. - PMC - PubMed

[2] Haraszti R.A., Roux L., Coles A.H., Turanov A.A., Alterman J.F., Echeverria D., Godinho B.M.D.C., Aronin N., Khvorova A. 5′-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo. Nucleic Acids Res. 2017;45:7581–7592. - PMC - PubMed

[3] Hassler M.R., Turanov A.A., Alterman J.F., Haraszti R.A., Coles A.H., Osborn M.F., Echeverria D., Nikan M., Salomon W.E., Roux L. Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo. Nucleic Acids Res. 2018;46:2185–2196. - PMC - PubMed

[4] Hassler M.R., Turanov A.A., Alterman J.F., Haraszti R.A., Coles A.H., Osborn M.F., Echeverria D., Nikan M., Salomon W.E., Roux L. Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo. Nucleic Acids Res. 2018;46:2185–2196. - PMC - PubMed

[5] Pasi K.J., Rangarajan S., Georgiev P., Mant T., Creagh M.D., Lissitchkov T., Bevan D., Austin S., Hay C.R., Hegemann I. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N. Engl. J. Med. 2017;377:819–828. - PubMed

[6] Pasi K.J., Rangarajan S., Georgiev P., Mant T., Creagh M.D., Lissitchkov T., Bevan D., Austin S., Hay C.R., Hegemann I. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N. Engl. J. Med. 2017;377:819–828. - PubMed

[7] Ray K.K., Landmesser U., Leiter L.A., Kallend D., Dufour R., Karakas M., Hall T., Troquay R.P., Turner T., Visseren F.L. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 2017;376:1430–1440. - PubMed

[8] Ray K.K., Landmesser U., Leiter L.A., Kallend D., Dufour R., Karakas M., Hall T., Troquay R.P., Turner T., Visseren F.L. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 2017;376:1430–1440. - PubMed

[9] Alterman J.F., Hall L.M., Coles A.H., Hassler M.R., Didiot M.-C., Chase K., Abraham J., Sottosanti E., Johnson E., Sapp E. Hydrophobically modified siRNAs silence Huntingtin mRNA in primary neurons and mouse brain. Mol. Ther. Nucleic Acids. 2015;4:e266. - PMC - PubMed

[10] Alterman J.F., Hall L.M., Coles A.H., Hassler M.R., Didiot M.-C., Chase K., Abraham J., Sottosanti E., Johnson E., Sapp E. Hydrophobically modified siRNAs silence Huntingtin mRNA in primary neurons and mouse brain. Mol. Ther. Nucleic Acids. 2015;4:e266. - PMC - PubMed

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Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier

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Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier

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