PEPTIDE-CONJUGATED MORPHOLINO OLIGOMERS FOR TREATMENT OF SPINAL MUSCULAR ATROPHY

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease and the first known genetic cause of infant mortality. It is caused by the homozygous mutations in the Survival Motor Neuron 1 (SMN1) gene, resulting in deficiency of functional SMN protein. The pathologic aspect of SMA is the progressive loss of Motor Neurons (MNs) in the ventral horn of the spinal cord, which causes the loss of muscle function, paralysis and premature death. A first drug for SMA, Nusinersen an antisense oligonucleotide (ASO) has been recently been approved. In fact, one of the most promising strategies in order to increase the SMN protein levels is the use of ASOs to redirect the splicing of the paralogous gene SMN2 to increase the production of the functional SMN protein. ASOs with different chemical structure are currently used in clinical trials, included the ASO with Morpholino (MO) chemistry studied in this work. MO oligomers are particularly suitable for in vivo applications thanks to their optimal safety and efficacy profile. One of the issues related to the administration of ASOs is that they are not able to cross the blood-brain barrier and they must be administered by relatively invasive intrathecal injection to reach the central nervous system (CNS). Therefore, we aimed to improve the efficacy of MO and to develop a non-invasive systemic delivery in an in vivo SMA murine model. First, to increase the efficacy of MO oligomers, we tested, both in vitro and in vivo, four new MO sequences targeting the region involved in the alternative splicing of SMN2 mRNA, called intronic splicing silencer N1 (ISS-N1). Simultaneously, to improve the delivery of MO to affected tissues, we conjugated it to cell-penetrating peptides (CPPs), an approach already used to enhance the cellular and tissue uptake and the pharmacological profile of ASOs, but never explored in SMA. We tested three different CPPs: Tat, R6, and (RXRRBR)2XB, which were linked to our already validated MO sequence [HSMN2Ex7D(-10,-34)]. We administered the conjugates in pre-symptomatic and symptomatic SMA mice to determine their therapeutic efficacy versus unconjugated-MO. We showed that optimization of the target sequence can enhance the splice-correction action of MO oligomers and that the effect of the co-administration of different MO sequences is superior to the delivery of a single sequence. Furthermore, we showed that CPP-MO compounds can up-regulate the SMN protein into the CNS of SMA7 transgenic murine models as well as their average lifespan and motor performance after local and systemic injection. Our data proved that the CPP-MO conjugates can be a suitable strategy to increase the cellular uptake after an intrathecal administration and to deliver MO oligomers to the CNS after a systemic injection. The presented results provided the basis for the selection of the most efficient CPPs and will set the stage for future clinical trials.