Abstract
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease affecting the upper and lower motor neurons in the motor cortex and spinal cord. Abnormal accumulation of mutant superoxide dismutase I (SOD1) in motor neurons is a pathological hallmark of some forms of the disease. We have shown that the orderly progression of the disease may be explained by misfolded SOD1 cell-to-cell propagation, which is reliant upon its active endogenous synthesis. Reducing the levels of SOD1 is therefore 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. In this study, calcium phosphate lipid coated nanoparticles (CaP-lipid NPs) were developed for delivery of SOD1 ASO to motor neurons. The most promising nanoparticle formulation (Ca/P ratio of 100:1), had a uniform spherical core–shell morphology with an average size of 30 nm, and surface charge (ζ-potential) of −4.86 mV. The encapsulation efficiency of ASO was 48% and stability studies found the particle to be stable over a period of 20 days. In vitro experiments demonstrated that the negatively charged ASO-loaded CaP-lipid NPs could effectively deliver SOD1-targeted ASO into a mouse motor neuron-like cell line (NSC-34) through endocytosis and significantly down-regulated SOD1 expression in HEK293 cells. The CaP-lipid NPs exhibited a pH-dependant dissociation, suggesting that that the acidification of lysosomes is the likely mechanism responsible for facilitating intracellular ASO release. To demonstrate tissue specific delivery and localization of these NPs we performed in vivo microinjections into zebrafish. Successful delivery of these NPs was confirmed for the zebrafish brain, the blood stream, and the spinal cord. These results suggest that CaP-lipid NPs could be an effective and safe delivery system for the improved delivery of SOD1 ASOs to motor neurons. Further in vivo evaluation in transgenic mouse models of SOD1 ALS are therefore warranted.
Highlights
Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that is associated with protein misfolding and aggregation
We used an encapsulation method for SOD1-Antisense oligonucleotides (ASOs) analogous to that reported by Tang et al where half of the ASO was mixed with the calcium and the phosphate solutions to yield a significantly greater EE than if the ASO was loaded into the calcium or phosphate phases alone (Tang et al, 2015) (Figure S1)
While there are no approved gene therapy products on the market for ALS, multiple oligonucleotide-based compounds are under development for the treatment of brain disorders by direct delivery inside the blood brain barrier (BBB)
Summary
Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that is associated with protein misfolding and aggregation. Due to its early discovery in 1993, the best-studied mutation associated with the familial form of ALS (fALS) is in the gene encoding copper/zinc superoxide dismutase (Cu/Zn SOD, SOD1) (Rosen et al, 1993). The correctly folded and active form of the SOD1 enzyme is obtained through several post-translational modifications such as the acquisition of zinc and copper ions, disulfide bond formation, and dimerization (Arnesano et al, 2004). Mutations that lead to the dissociation of metal ions, and reduction of the intramolecular disulfide bond are known to decrease its conformational stability and promote misfolding. It is well accepted that mutations in SOD1 cause ALS through a toxic gain of function rather than a loss of its native function. Over 180 mutations in the SOD1 gene that impact upon its structural stability have been identified Over 180 mutations in the SOD1 gene that impact upon its structural stability have been identified (http://alsod.iop. kcl.ac.uk/Overview/gene.aspx?gene_id=SOD1)
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