Universität zu Köln

Muinos-Buehl, A. (2023). Development of antisense oligonucleotide combinatorial therapies for spinal muscular atrophy (SMA). PhD thesis, Universität zu Köln.

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Abstract

Spinal muscular atrophy (SMA) is a devastating genetically inherited neuromuscular disorder characterized by the progressive loss of α-motor neurons (MNs) in the anterior horn of the spinal cord, leading to muscle atrophy and weakness. Without treatment, SMA is the leading genetic cause of infant death. Although SMA is caused by homozygous mutations in survival motor neuron 1 (SMN1) gene, the disease severity is mainly determined by SMN2 copy number, an almost identical gene that produces ~10% correctly spliced full length SMN transcripts. Recently, three FDA- and EMA-approved therapies that either increase correctly spliced SMN2 transcripts (nusinersen - antisense oligonucleotide, ASO - and risdiplam - small molecule - ) or replace SMN1 gene (onasemnogen abeparvovec-xioi) have revolutionized the clinical outcome of SMA patients. However, clinical and scientific evidence emphasize the importance of a presymptomatic treatment in order achieve a significant therapeutic outcome. Moreover, there is a significant number of patients that do not respond to the SMN-enhancing treatments. Indeed, for severely affected SMA individuals carrying only two SMN2 copies even a presymptomatic therapy might be insufficient to fully counteract disease development. Therefore, SMN-independent compounds supporting SMN-dependent therapies represent a promising therapeutic approach. In this regard, genetic modifiers such as PLS3, NCALD or CHP1 have proved to act protective against SMA across species. Therefore, the aims of this work are the following: 1) To test a combinatorial therapy using SMN-ASO and Chp1-ASOs in SMA mice, 2) To develop an efficient MN differentiation protocol from healthy and SMA hiPSCs to use as a platform for compound screening (ASOs), 3) To test the long-term effect of the combinatorial therapy based on SMN-ASO and Ncald-ASO. In the first part of this work, we focused on the combinatorial treatment enhancing SMN protein levels and reducing CHP1 protein amount. CHP1, calcineurin-like EF-hand protein 1, is an interacting partner of PLS3, a strong modifier of SMA, and acts as a negative regulator of neurite outgrowth and endocytosis. Importantly, a significant amelioration of SMA disease hallmarks was observed in a severely affected SMA mouse model carrying a mutant Chp1 allele when combined with a suboptimal dose of SMN-ASO treatment. In this work, we aimed to pharmacologically reduce CHP1 levels in an ASO-based combinatorial therapy targeting SMN and Chp1 in SMA mice. Notably, Chp1 modulation represents a major challenge since its reduction to ~50% showed an amelioration of SMA pathology, while the downregulation below those levels is detrimental and prompts cerebellar ataxia characterized by Purkinje neuron loss. Hereby, efficacy and tolerability studies in neonatal wild type mice determined that a single injection of 30 µg Chp1-ASO4 in the CNS is a safe dosage that significantly reduced CHP1 levels to about 50% at postnatal day (PND)14 in the brain and the spinal cord. Unfortunately, neither electrophysiological predictors such as compound muscle action potential XVI | S U M M A R Y (CMAP) or motor unit number estimation (MUNE) nor morphological properties of the neuromuscular junctions (NMJ), the spinal cord or the tibialis anterior muscle were ameliorated in SMA mice treated with Chp1-ASO4 compared to CTRL-ASO at PND21. Unexpectedly, CHP1 levels were not reduced at 4- weeks post injection, indicating a rather short-term effect and stability of the ASO. Next, we re�administrated Chp1-ASO4 by i.c.v. bolus injection at PND28. However, no significant improvement of SMA hallmarks was observed at 2 month-of-age. Taken together, in contrast to the protective effect of the genetically-induced Chp1 reduction on SMA, combinatorial therapy based on Chp1- and SMN�ASOs failed to significantly ameliorate the SMA pathology in mice. The short-term stability of Chp1- ASOs compared to SMN-ASO suggests that further optimization of the ASO may be required to fully explore the combination of treatments. The second part of this work focused on the long-term combinatorial treatment targeting SMN upregulation and Ncald reduction. NCALD, neurocalcin delta, was identified as an SMA protective modifier in a SMA discordant family, where asymptomatic individuals exhibited a 4-5 fold NCALD reduction. NCALD is a calcium sensor protein that negatively regulates clathrin-mediated endocytosis and axonal outgrowth. NCALD reduction improves impaired endocytosis and ameliorates SMA pathology across species. Moreover, previous work carried out in our research group demonstrated that NCALD pharmacological reduction using Ncald-ASO in a low-dose SMN-ASO treated severe SMA mouse model, significantly improved electrophysiological and morphological pathology hallmarks at PND21. However, at 3 months of age SMA mice exhibited amelioration of motoric abilities solely. In the present work, we aimed to study the long-term effect of Ncald-ASO by re-injecting it via i.c.v. bolus administration at PND28. In parallel, we aimed to test the therapeutic effect of human NCALD-ASOs in MNs differentiated from patient-derived hiPSCs. First, 500µg Ncald-ASO administered via i.c.v. bolus injection at PND28 in wild type mice showed a good tolerability of the procedure and a significant reduction of NCALD in the brain and the spinal cord after two weeks. Importantly, Ncald-ASO re�injection prolonged the amelioration of electrophysiological parameters and rescued denervation defects. Moreover, human NCALD-ASO69 significantly downregulated NCALD levels in hiPSC derived MNs from healthy and SMA type I individuals. In addition, growth cone morphology and spontaneous neuronal activity were improved upon NCALD-ASO69 treatment. These data strongly supports the therapeutic role of NCALD in SMA and the importance of maintaining its reduction in order to achieve better clinical outcomes

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Muinos-Buehl, A. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-706731
Date:	2023
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Zentrum für Molekulare Medizin
Subjects:	Natural sciences and mathematics Medical sciences Medicine
Uncontrolled Keywords:	Keywords Language spinal muscular atrophy UNSPECIFIED antisense oligonucleotide UNSPECIFIED rare diseases UNSPECIFIED neuromuscular disease UNSPECIFIED motor neurons UNSPECIFIED
Date of oral exam:	24 February 2023
Referee:	Name Academic Title Rugarli, Elena Prof. Vilchez, David Prof.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/70673