Morpholino–RNA duplex exhibits robust, sustained, and safe steric-block antisense activity by intracerebroventricular and intrathecal injection

Intrathecal injection of steric-block antisense oligonucleotides (ASOs) provides therapeutic opportunities for targeting the central nervous system (CNS). Phosphorothioate backbone with 2'-O-methoxyethyl (MOE) is widely used in ASO drugs, with concerns about toxicity and potency. Phosphorodiamidate morpholino oligomer (PMO) is another chemistry with a favorable safety profile but less persistent effect due to its ineffective cellular uptake and poor binding affinity to serum proteins. Here, a cholesterol-conjugated PMO/RNA heteroduplex oligonucleotide (Chol-HDO[PMO]) outperformed conventional single-stranded ASOs, achieving up to a 16-fold increase in potency without detectable toxicity and maintaining stronger ASO activity throughout the observation period. Chol-HDO[PMO] showed enhanced antisense activity in both splice modulation and translation inhibition. Chol-HDO[PMO] successfully alleviated the CNS symptoms in a Duchenne muscular dystrophy mouse model. In contrast, Chol-HDO[MOE] failed to enhance tissue delivery or potency. The efficient tissue delivery relied on interactions with CSF lipoproteins, and binding affinity was determined by heteroduplex oligonucleotide chemistry. Collectively, Chol-HDO[PMO] may expand the therapeutic potential of steric-block ASOs targeting the CNS.