Peptide-conjugated Morpholino Oligomers for treatment of Spinal Muscular Atrophy

The use of Antisense oligonucleotide(ASO) represents a promising treatment for Spinal Muscular Atrophy (SMA) by its ability to increase the production of a functional SMN protein and rescue the phenotype in SMA animal models. However, there are several hurdles to overcome. To increase the cellular and tissue uptake and pharmacological profile of Morpholino Oligomers (MO), an ASO variants, one possible strategy is the conjugation with cell-penetrating peptides (CPPs). In this study we investigated the efficacy of different CPPs linked to our validated MO sequence (Tat, R6, r6 and (RXRRBR)2XB) and of novel MOs sequences in in vitro and in vivo SMA models. In vitro, we nucleofected induced pluripotent stem cells (iPSCs) derived from SMA patients with the four MOs. The treatment with MO B and D, and in particular their combination, showed a consistent increase of SMN protein levels and a significant upregulation of SMN GEMS in the cell nuclei. The same increment was obtained in vivo in the SMA7 mouse model. Moreover, we administered our alreadyvalidated MO sequence (MO-10-34) conjugated with four CPPsin a small pilot group of pre-symptomatic SMA mice, using the protocol already established for unconjugated MO (Nizzardo et al., 2014).The best conjugated was selected for next studies in presymptomatic and symptomatic SMA mice to assess its therapeutic potential. We will assess the feasibility of this strategy to: 1) cross the blood brain barrier, allowing MO non-invasive systemic delivery, and 2) treat the disease in a symptomatic phase, expanding the therapeutic window.