Abstract Details

Title In-vitro Efficacy and In-vivo Biodistribution Studies of an Antisense Oligonucleotide Targeting Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation

Topic Child Neurology and Developmental Neurology

Presentation(s) S37 - Emerging Therapies in Child Neurology (5:18 PM-5:30 PM)

Poster/Presentation
Number 010

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.

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).

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.

Authors/Disclosures
Manouchehr Amanat PRESENTER	Mr. Amanat has nothing to disclose.
Christina Nemeth Mertz (Kennedy Krieger Institute)	No disclosure on file
Ines Garofolo (Kennedy Krieger Institute)	No disclosure on file
Amena S. Fine, MD, PhD (Kennedy Krieger Institute)	The institution of Dr. Fine has received research support from NIH.
Seyed A. Fatemi, MD, MBA (Kennedy Krieger Institute, Johns Hopkins Medical Institute)	Dr. Fatemi has received personal compensation in the range of $10,000-$49,999 for serving as an Expert Witness for RCO law. The institution of Dr. Fatemi has received research support from Minoryx. The institution of Dr. Fatemi has received research support from Viking Therapeutics. The institution of Dr. Fatemi has received research support from Swanbio Therapeutics . The institution of Dr. Fatemi has received research support from Autobahn Therapeutics. The institution of Dr. Fatemi has received research support from Nuvelution. The institution of Dr. Fatemi has received research support from NIH. The institution of Dr. Fatemi has received research support from A Cure for Ellie Foundation. The institution of Dr. Fatemi has received research support from Brian's Hope Foundation . Dr. Fatemi has received intellectual property interests from a discovery or technology relating to health care. Dr. Fatemi has a non-compensated relationship as a Director (Board member) with ALDConnect that is relevant to AAN interests or activities.