Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds

Abstract

Niemann-Pick disease type C1 (NPC1) is a rare autosomal recessive lysosomal storage disease primarily caused by mutations in NPC1. NPC1 is characterized by abnormal accumulation of unesterified cholesterol and glycolipids in late endosomes and lysosomes. Common signs include neonatal jaundice, hepatosplenomegaly, cerebellar ataxia, seizures and cognitive decline. Both mouse and feline models of NPC1 mimic the disease progression in humans and have been used in preclinical studies of 2-hydroxypropyl-β-cyclodextrin (2HPβCD; VTS-270), a drug that appeared to slow neurological progression in a Phase 1/2 clinical trial. However, there remains a need to identify additional therapeutic agents. High-throughput drug screens have been useful in identifying potential therapeutic compounds; however, current preclinical testing is time and labor intensive. Thus, development of a high-capacity in vivo platform suitable for screening candidate drugs/compounds would be valuable for compound optimization and prioritizing subsequent in vivo testing. Here, we generated and characterize two zebrafish npc1-null mutants using CRISPR/Cas9-mediated gene targeting. The npc1 mutants model both the early liver and later neurological disease phenotypes of NPC1. LysoTracker staining of npc1 mutant larvae was notable for intense staining of lateral line neuromasts, thus providing a robust in vivo screen for lysosomal storage. As a proof of principle, we were able to show that treatment of the npc1 mutant larvae with 2HPβCD significantly reduced neuromast LysoTracker staining. These data demonstrate the potential value of using this zebrafish NPC1 model for efficient and rapid in vivo optimization and screening of potential therapeutic compounds..

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

“…[205][206][207] Other studies have utilized endonucleases to generate different kinds of immunodeficient animal models that were previously unable to be established due to a lack of effective genetic modification. [207][208][209][210] As a result of engineered nucleasemediated editing of genomic modifications, other animal disease models have been developed, simulating Rett syndrome, 211 hereditary deafness, 212 Wilson disease, 213 Laron syndrome, 214 Niemann-Pick disease, 215 Netherton syndrome, 216 and so on. Advances in genome editing technologies will further expand the application of animal models in disease mechanism research and treatment development.…”

Using CRISPR/Cas9 to model human liver disease

“…Two groups independently reported zebrafish models for Niemann-Pick disease type C1 (NPC1). 65,66 NPC is a rare autosomal recessive disease caused by the accumulation of cholesterol in late endosomes/lysosomes. 67 Currently, there are no effective therapies for NPC.…”

“…Common manifestations of NPC1 include hepatomegaly and severe cirrhosis, along with progressive neurodegeneration. 68 Tseng et al 65 and Lin et al 66 utilised embryo injection with sgRNA and Cas9 mRNA to generate npc1 knockout zebrafish. Deficiency of npc1 recapitulated both the early-onset hepatic disease and the later neurological disease observed in patients with NPC1, namely, cholesterol accumulation in the liver and symptoms of ataxia.…”

“…In addition, a robust increase in in vivo LysoTracker Red staining was observed in npc1 mutants as early as 3 days post fertilisation. 65 The ability to rapidly access pathophysiological readouts highlights the advantage of using CRISPR/Cas9-edited animals to accelerate large-scale phenotypic screening, which could be used to evaluate candidate drugs and compounds for new therapies.…”

Swimming in Deep Water: Zebrafish Modeling of Complicated Forms of Hereditary Spastic Paraplegia and Spastic Ataxia

“…Mutations in this gene are responsible for 95% of cases of Niemann-Pick disease, a rare AR lysosomal storage disorder (Torres et al, 2017) with heterogeneous symptoms that include jaundice, hepatosplenomegaly, ataxia, spasticity, and intellectual decline leading to dementia (Schwend et al, 2011;Louwette et al, 2013;Lin et al, 2018;Tseng et al, 2018). The transmembrane protein NPC1 is implicated in the retrograde transport of cholesterol and glycolipids from lysosomes, and mutated NPC1 causes an accumulation of these substances in lysosomes, leading to onset of variable neurological manifestations (Lin et al, 2018;Tseng et al, 2018). The first studies of this gene in zebrafish were conducted using MOs, but morphants were characterized by premature death, before 2 dpf (Schwend et al, 2011) or at around 5 dpf when higher doses of MO were used (Louwette et al, 2013).…”