DG9-conjugated morpholino rescues phenotype in SMA mice by reaching the CNS via a subcutaneous administration

Abstract

Antisense oligonucleotide (AO)-mediated therapy is a promising strategy to treat several neurological diseases including spinal muscular atrophy (SMA). However, limited delivery to the central nervous system (CNS) with AOs administered intravenously or subcutaneously is a major challenge. Here we demonstrate a single subcutaneous administration of cell-penetrating peptide DG9 conjugated to an AO called phosphorodiamidate morpholino oligomers (PMOs) reaches the CNS and significantly prolonged the median survival compared to unconjugated PMO and R6G-PMO in a severe SMA mouse model. Treated mice exhibited significantly higher expression of full-length SMN2 expression (FL-SMN2) in both the CNS and systemic tissues compared to non-treated and unmodified AO-treated mice. The treatment ameliorated the atrophic musculature and improved breathing function accompanied by improved muscle strength and innervation at the neuromuscular junction with no signs of apparent toxicity. We also demonstrated DG9-conjugated PMO localizes in nuclei in the spinal cord and brain after subcutaneous injections. Our data identify DG9 peptide conjugation as a powerful way to improve the efficacy of AO-mediated splice modulation. Finally, DG9-PMO is a promising therapeutic option to treat SMA and other neurological diseases, overcoming the necessity for intrathecal injections and treating body-wide tissues without apparent toxicity.

Potential of Cell-Penetrating Peptide-Conjugated Antisense Oligonucleotides for the Treatment of SMA

“…Conjugated to an 18-mer PMO with the same sequence as Nusinersen, the DG9 CPP was subcutaneously administered to SMA Taiwanese mice at PD-0. Compared to R6G-PMOs, used as a benchmark control peptide PMO conjugate, as well as unconjugated PMOs and 2'-MOEs, DG9-PMO treatment yielded the most improved muscle strength and improved median life span, increasing it from 12 days for unconjugated PMOs to 58 days for DG9-PMO treated mice [9]. To assess DG9-PMO uptake into the CNS at a more advanced age, a milder SMA mouse model received subcutaneous injections of DG9-PMO at PD-5.…”

Cell-Penetrating Peptides-Mediated Therapeutic Agents Delivery into the Central Nervous System

“…This coupling not only enhances the stability and activity of nucleic acids but also facilitates their efficient delivery. , In addition, CPPs coupled with nucleic acids have the ability to selectively target specific pathways or targets involved in CNS diseases, such as amyloid, tau proteins, and nerve growth factors, resulting in specific and highly effective therapeutic effects . Tejal et al employed CPPs DG-9 and phosphorodiamidate morpholino oligomer (PMO) to bind, which were administered subcutaneously in a mouse model of severe spinal muscular atrophy (SMA). As shown in Figure A, the conjugate entered the CNS and exhibited a substantial increase in the survival duration of the mice, along with notable improvements in their motor performance.…”

“…Reprinted with permission. Copyright 2023, Aslesh et al (B) Regulation of Th17 cell differentiation and alleviation of EAE by AP-ctCTLA-4 reprinted with permission from ref Reprinted with permission. Copyright 2021, Won-Ju Kim et al (C) Brain delivery of insulin boosted by intranasal coadministration with cell-penetrating peptides.…”

Potential of Cell-Penetrating Peptide-Conjugated Antisense Oligonucleotides for the Treatment of SMA

“…In 2023, Aslesh et al investigated the potential of the DG9 peptide, derived from the human polyhomeotic 1 homolog transcription factor, as a PMO conjugate in SMA mice [ 9 ]. Conjugated to an 18-mer PMO with the same sequence as Nusinersen, the DG9 CPP was subcutaneously administered to SMA Taiwanese mice at PD-0.…”

“…Among these, cell-penetrating peptides (CPPs) emerged as a promising small-molecule carrier. When directly conjugated to neutrally charged ASOs, CPPs were seen to not only improve ASO cellular uptake but also facilitate delivery into the CNS following system injection in SMA mice [ 5 , 6 , 7 , 8 , 9 ]. Despite the evident potential of CPPs to enhance ASO delivery, they have yet to be FDA-approved as a carrier due to concerns regarding their toxicity and immunogenicity [ 10 ].…”