Using calcium phosphate lipid-coated nanoparticles to deliver antisense oligonucleotides to motor neurons in motor neurone disease

Vine KL1,2, Watson C1,2, Chen L1,2, Saunders D3, Morsch M4, Chung R4, Cole N4 and Yerbury JJ1,2

  1. School of Biological Sciences, Faculty Science, Medicine and Health, University of Wollongong, NSW, 2522, Australia.
  2. Illawarra Health and Medical Research Institute, Wollongong, NSW, 2522, Australia.
  3. School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.
  4. Department of Biomedical Sciences, Macquarie University, Sydney, NSW, 2113, Australia.

Introduction: Abnormal accumulation of mutant superoxide dismutase I (SOD1) in motor neurons is a pathological hallmark of some forms of ALS. Considering that SOD1 can propagate from cell-to-cell in a prion-like fashion, potentially contributing to the orderly progression of the disease [1], reducing levels of SOD1 is a promising therapeutic approach. Antisense oligonucleotides (ASOs) can efficiently silence proteins with gain-of-function mutations. However, naked ASOs have a short circulation half-life and are unable to cross the blood brain barrier (BBB) warranting the use of a drug carrier for effective delivery. We therefore aimed to improve the delivery of gene therapies to motor neurons in the context of ALS [2], using solid core calcium phosphate lipid-coated nanoparticles (CaP-lipid NPs), encapsulating an ASO directed to SOD1. Here we report the manufacture and biophysical characterization of CaP-lipid NPs that encapsulate a SOD1 ASO and describe their in vitro uptake and in vivo distribution in a zebrafish model. Methods: CaP-lipid NPs were prepared as described in our paper [3] and characterized for and size (nm), zeta potential (mV), polydispersity index (PDI), encapsulation effiencicy (%) and particle concentration. The in vitro cellular uptake and gene silencing were assessed by confocal microscopy and Western blotting, respectively. The in vivo distribution was assessed using a well-established ALS zebrafish model [4]. Results: Specifically, we demonstrated that the delivery of CaP-lipid NPs is efficacious in a motor-neuron-like established cell line (NSC-34) and in primary motor neuron cultures isolated from murine spinal cords [5] by confocal microscopy. Significant down-regulation of SOD1 protein expression was confirmed by immunoblotting following the delivery of SOD1 ASO-loaded CaP-lipid NPs. We also describe for the first time nanoparticle distribution in the brain, spinal cord and blood circulation of zebrafish, a powerful experimental vertebrate model for studying ALS. Conclusions: Our results suggest that CaP-lipid NPs could be an effective and safe system for the improved delivery of SOD1 ASOs to affected motor neurons in ALS. Acknowledgements This work has been supported by the Motor Neuron Disease Research Institute of Australia (2016-2017) and the US Department of Defence (AL150057; 2016-2018). References [1] Grad et al. 2014 PNAS, 111 (9), 3620-3625 [2] Foust et al. 2013 Molecular Therapy, 21(12): 2148–2159 [3] Chen et al. 2017 Frontiers in Neuroscience, 11:476 [4] Morsch et al. 2017 Journal of Visualized Experiments, (120): 54983 [5] Kieran & Greensmith 2004 Neuroscience (125): 427-439.