In-vitro Efficacy and In-vivo Biodistribution Studies of an Antisense Oligonucleotide Targeting Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation
Manouchehr Amanat1, Christina Nemeth2, Ines Garofolo2, Amena Fine3, Ali Fatemi1
1Neurology, Johns Hopkins School of Medicine, 2Neuroscience, 3Neurology, Kennedy Krieger Institute
Objective:
This study aimed to explore the efficacy of antisense oligonucleotides (ASOs) to modify DARS2 gene expression in leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL).
Background:
LBSL is an ultra-rare neurodevelopmental disorder, caused by compound heterozygote variants in DARS2 gene. Most patients have one pathogenic variant in the second intron, affecting the splicing of exon 3. It is hypothesized that increasing DARS2 exon 3 inclusion may restore protein function.
Design/Methods:
We designed fifteen ASOs to target intronic splicing silencers located upstream of exon 3. Induced pluripotent stem cells (iPSCs) were generated from the blood cells of three LBSL patients and one healthy individual. One ASO with the highest efficacy to restore DARS2 exon 3 expression in iPSCs was selected. This ASO was tested at 50 nM and 100 nM on iPSC-derived neural progenitor cells (NPCs) to measure DARS2 exon 3 expression and neurite outgrowth. Mitochondrial function of the iPSC-derived neurons was tested through measuring lactate levels in the cultured media and oxygen consumption rate (OCR; The Seahorse Assay). In a pilot experiment, 40 µg of a tagged ASO was delivered into three wild-type mice through intracerebroventricular injection to assess ASO biodistribution.
Results:
Our designed ASO increased DARS2 exon 3 expression in all LBSL iPSC-derived NPCs after 48 hours in a dose-dependent manner. Higher neurite outgrowth per cell was noted in treated NPCs compared to untreated ones after 72 hours. Additionally, lactate levels significantly reduced and maximum OCR increased in treated LBSL neurons, compared to untreated cells after one week of ASO administration. The biodistribution study showed that ASO signal was detected within neurons of the hippocampus and cortex one week after injection.
Conclusions:
ASO therapy holds promise for restoring gene expression, as well as improving neurite outgrowth and mitochondrial function in iPSC-derived NPCs and neurons obtained from LBSL patients.