Project Overview

“Antisense oligonucleotides (ASOs) are synthetic single-stranded DNA/RNA that are useful in the targeting of mRNA sequences. Their versatility allows them to enhance potency, longevity and bioavailability, making them a popular therapeutic for neurodegenerative diseases and a leading approach in clinical trials for Huntington disease (HD). However, the blood-brain barrier (BBB) limits ASO effectiveness in neurodegenerative diseases, and are often administered intrathecally to the spinal cord. This is an invasive method with limited ASO delivery to deep brain regions, like the striatum.

To overcome this, nanodiscs (created by Dr. Dale Martin) can be used to efficiently distribute ASOs throughout the brain, as shown in a recent study on lowering mutant huntingtin (mHTT) in HD models. These nanodiscs are formed with apolipoprotein A-I (apoA-I), which creates a nascent high-density lipoprotein (nHDL) disc. This nHDL disc shape is stabilized with a K133C mutation, which allows the nHDL to bypass endogenous pathways to deliver more ASO to the brain. However, current nandiscs are limited to binding to the two apoA-I proteins in each disc.

My project will focus on optimizing nanodisc-ASO composition to improve ASO delivery throughout the brain. Identifying the most efficient composition will help advance delivery methods in treating HD and various neurodegenerative diseases. It will offer patients less invasive treatment methods with the option of intravenous or intranasal administration while still persevering treatment efficiency. Studies show that intravenous tail injections deliver ASOs to all regions of the brain, and intranasal injections possess multiple routes for delivery to various brain tissues.

To start, I will use a gapmer ASO targeting mHTT, modified to allow conjugation to fatty acids. I recently optimized the purification of His-tagged human apoA-I. Currently, nanodiscs are generated by combining apoA-I with dimyristoylphosphatidylcholine (DMPC).

I aim to increase ASO delivery by increasing the ratio of nHDL lipids to ASO interactions, so that more ASOs can bind to the disc surface instead of the apoA-I proteins. This can be accomplished by conjugating fatty acids to the ASO, allowing direct linkage to nanodisc phospholipids. Alternatively, we can increase the positive charge of phospholipids to promote electrostatic interactions with negatively charged ASOs, which can be done using the DMPC derivative, dimyristoyltrimethylammonium propane (DMTAP). Preliminary data indicates that nanodiscs containing DMTAP are more readily taken up in heterologous/primary neuronal cell culture.

To deduce which method best increases ASO delivery, the following will be tested in HEK293T cells:
– (1) Nanodisc from DMPC with [a] No ASO, [b] ASO, [c] Lipidated ASO
– (2) Nanodisc from DMPC & DMTAP with [a] No ASO, [b] ASO, [c] Lipidated ASO

The most promising apoA-I nanodisc + ASO composition will be similarly tested in primary neuronal cells derived from YAC128 mice, to ensure enhanced delivery in disease-relevant cells. I will confirm their uptake by detecting ApoA-I by western blot complemented with near super resolution confocal microscopy.

Optimizing nanodisc composition will increase ASO delivery to the brain. Ultimately, this can positively impact the development of therapies that cross the BBB, decrease invasiveness, and can be used for various neurological conditions. “

Principal Investigator

Yusra Kureshi , University of Waterloo

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