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Intravenously Administered Novel Liposomes, DCL64, Deliver Oligonucleotides to Cerebellar Purkinje Cells

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Abstract

Cerebellar Purkinje cells (PCs) show conspicuous damages in many ataxic disorders. Targeted delivery of short nucleic acids, such as antisense oligonucleotides, to PCs may be a potential treatment for ataxic disorders, especially spinocerebellar ataxias (SCAs), which are mostly caused by a gain of toxic function of the mutant RNA or protein. However, oligonucleotides do not cross the blood-brain barrier (BBB), necessitating direct delivery into the central nervous system (CNS) through intra-thecal, intra-cisternal, intra-cerebral ventricular, or stereotactic parenchymal administration. We have developed a novel liposome (100 to 200 nm in diameter) formulation, DCL64, composed of dipalmitoyl-phosphatidylcholine, cholesterol, and poloxamer L64, which incorporates oligonucleotides efficiently (≥ 70%). Confocal microscopy showed that DCL64 was selectively taken up by brain microvascular endothelial cells by interacting with low-density lipoprotein receptor (LDLr) family members on cell surface, but not with other types of lipid receptors such as caveolin or scavenger receptor class B type 1. LDLr family members are implicated in brain microvascular endothelial cell endocytosis/transcytosis, and are abundantly localized on cerebellar PCs. Intravenous administration of DCL64 in normal mice showed distribution of oligonucleotides to the brain, preferentially in PCs. Mice that received DCL64 showed no adverse effect on hematological, hepatic, and renal functions in blood tests, and no histopathological abnormalities in major organs. These studies suggest that DCL64 delivers oligonucleotides to PCs across the BBB via intravenous injection with no detectable adverse effects. This property potentially makes DCL64 particularly attractive as a delivery vehicle in treatments of SCAs.

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Acknowledgments

We are grateful to the technical support provided by University of Florida (UF) ICBR Cytometry Core, ICBR Electron Microscopy Core, MBI-UF Cell & Tissue Analysis Core, UF Molecular Pathology Core, and UF Animal Care Services.

Funding

This study was supported by UF Opportunity Grant to ATA and TA.

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Author notes

  1. Ana Tari Ashizawa

    Present address: Bio-Path Holdings, Inc., Bellaire, TX, USA

Authors and Affiliations

  1. McKnight Brain Institute, University of Florida, Gainesville, FL, USA

    Ana Tari Ashizawa, Jenny Holt, Kelsey Faust, Weier Liu & Tetsuo Ashizawa

  2. Department of Neuroscience, University of Florida, Gainesville, FL, USA

    Ana Tari Ashizawa, Kelsey Faust & Weier Liu

  3. Department of Neurology, University of Florida, Gainesville, FL, USA

    Jenny Holt & Tetsuo Ashizawa

  4. Stanley H. Appel Department of Neurology, Houston Methodist Research Institute, 6670 Bertner Avenue, R11-117, Houston, TX, 77030, USA

    Anjana Tiwari, Nan Zhang & Tetsuo Ashizawa

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  1. Ana Tari Ashizawa
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Corresponding author

Correspondence to Tetsuo Ashizawa.

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Conflict of Interest

ATA is an employee and stock holder of Bio-Path Holdings, Inc. TA is Adjunct Professor of Baylor College of Medicine, and receives grants from the NIH (R01 NS083564), the National Ataxia Foundation (NAF), the Myotonic Dystrophy Foundation (MDF), and the Marigold Foundation, and has been supported by Biohaven Pharmaceuticals and Ionis Pharmaceuticals for clinical trials of their drugs, and by Pacific Biosciences for symposium honoraria. TA also serves on the Medical and Research Advisory Board of the NAF, and the Scientific Advisory Board of the MDF. ATA and TA filed a US patent application (Serial # 14/390,584).

Research Involving Animals

The animal research in this work was approved by the UF Institutional Animal Care and Use Committee (IACUC #201307878).

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This work does not involve human subject research.

Electronic Supplementary Materials

Supplementary Figure 1

Caveolin-1 did not affect DCL64 liposomes binding to BMECs. BMECs were incubated in the absence (left panel) or presence (right panel) of 1.0 μg of peptides specific for caveolin-1 (Abcam) for 1 h before incubated with 0.5 μg of fluorescent liposomal oligonucleotides [red color] for an additional hour. Cells were fixed and counterstained with DAPI. (PNG 759 kb)

High resolution image (TIF 1501 kb)

Supplementary Figure 2

Intravenous injection of DCL64 liposomes led to oligonucleotide accumulation in PCs. Mice were injected with fluorescent DCL64 and euthanized 4 h postinjection. (Left) Calbindin staining of Purkinje cells [green color]; (Right) Detection of red fluorescent oligonucleotide in cerebellar Purkinje cells and dendrites in the molecular layer. (PNG 1629 kb)

High resolution image (TIF 5319 kb)

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Ashizawa, A.T., Holt, J., Faust, K. et al. Intravenously Administered Novel Liposomes, DCL64, Deliver Oligonucleotides to Cerebellar Purkinje Cells. Cerebellum 18, 99–108 (2019). https://doi.org/10.1007/s12311-018-0961-2

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