This is a genuine translational project in which we aim to develop a combinatorial therapy for SMA. It is based on our newly discovered protective modifier CHP1; we have shown that genetically induced Chp1 downregulation together with low dose SMN-ASOs ameliorates SMA deficits in a severe SMA mouse model and in zebrafish (1).
Here we aim to use the most efficient and least toxic Chp1-ASOs in two different preclinical randomized and blinded studies in SMA mice. Moreover, human CHP1-ASOs will be tested in motoneurons differentiated from IPSCs paving the ground for a therapy in humans.
Spinal muscular atrophy (SMA) is the leading genetic cause of early childhood lethality. Mutations in the SMN1 gene cause functional loss of a-motoneurons in the spinal cord mainly disturbing development and maturation of neuromuscular junctions. Impaired synaptic transmission causes muscle weakness and atrophy of proximal voluntary muscles.
The disease severity inversely correlates with the number of SMN2 copy genes –usually varying between 1-4 copies– each producing only 10% correctly spliced RNA and protein. The SMN2 copies represent the main target for SMA therapy. Nusinersen, the first SMN antisense oligonucleotides (ASOs) that restore correctly spliced SMN2 transcripts have been recently FDA- and EMA-approved for SMA therapy. While nusinersen show encouraging results, for the majority of patients, for patients who carry only two SMN2copies, the ASO-induced SMN elevation seems to be insufficient to fully counteract motoneuron dysfunction.
In the past few years, we identified two SMA protective modifier, PLS3 and NCALD, in asymptomatic SMN1-deleted individuals and verified the protective potency in various genetically modified SMA animal models (1-7). Moreover, we identified two further SMA protective modifier, CORO1C (Coronin 1C) and CHP1 (Calcineurin-like EF-hand protein 1) that interacts with PLS3 (1; 4). While PLS3 and CORO1C are F-actin binding and bundling proteins and act SMA protective upon overexpression, CHP1 and NCALD are calcium sensors and act protective by downregulation (1-7. Moreover, all four modifiers restore impaired endocytosis in SMA (1, 4, 5).
Here we aim to develop a combinatorial SMA therapy based on our novel SMA-protective modifier, CHP1, together with low-dose SMN-ASOs. A full battery of murine and human CHP1-ASOs have been designed for our study by IONIS Pharmaceuticals. The three best ASOs are currently tested in our lab for lowest toxicity and highest efficiency in SMA mice. In parallel human CHP1-ASOs are tested in MN-derived from iPSC from SMA and control individuals. Lastly, we aim to better understand the function and cellular mechanism contributing to the protective effect of CHP1. Therefore, a full batch of highly interesting novel interacting partners have been identified by proteome analysis and will be studied on biochemical, cellular and functional level.
The four aims of the project are
Combinatorial therapy using Chp1 + SMN-ASOs in a severely-affected SMA mouse model
Repetitive Chp1-ASO injection plus a single low-dose SMN-ASO injection in a severely-affected SMA mouse model
Develop and study human CHP1-ASOs in motoneurons and neuromuscular junctions differentiated from iPSCs
Molecular and cellular analysis of CHP1 downregulation in motoneurons and neuronal cells
We expect to set the ground for a novel efficient combinatorial SMA therapy targeting both a SMN-independent and SMN-dependent pathway and to further unveil the underlying protective pathway of CHP1.
Our recent work based on yeast-two-hybrid screens, identified calcineurin-like EF-hand protein 1 (CHP1) as a novel interacting partner of PLS3. Independent co-immunoprecipitation and pull-down experiments proved a direct interaction between CHP1 and PLS3. CHP1 is ubiquitously expressed, but particularly abundant in the neuronal tissue. CHP1 is a negative regulator of calcineurin, the most important phosphatase dephosphorylating the dephosphins involved in endocytosis.
Most importantly, we show that low-dose SMN-ASO-treated SMA mice and reduced CHP1 levels due to a heterozygous splice site mutation in Chp1vac/wt (vacillator variant), ameliorates the SMA phenotype as compared to only low-dose SMN-ASO-treated SMA mice (1). Thus, CHP1 is a highly exciting novel protective modifier, which not only interacts with PLS3 but is a calcium sensor, similar to NCALD, and acts SMA protective by downregulation, which can be achieved by specific ASO treatment.
Novelty and clinical relevance: This study aims to test a combinatorial therapy in SMA by using the power of protective genetic SMA modifiers. Here, we aim to restore motor neuron function using Chp1-ASOs (SMN-independent acting pathway) in combination with SMN-dependent ASOs (nusinersen) in a severely-affected SMA mouse model (8). Moreover, the development and testing of human CHP1-ASOs will be a next step towards developing a combinatorial therapy in humans. We will analyze the impact of human CHP1-ASOs on motor neurons differentiated from inducible pluripotent stem cells derived from SMA and control individuals. Lastly, by using Co-IP and mass spectrometry from wildtype versus CHP1vac/vacspinal cord we identified a plethora of highly interesting proteins that might further help to unveil the molecular and cellular pathway underlying SMA protection by CHP1 reduction.
We expect to set the ground for a novel efficient combinatorial therapy targeting both a SMN-independent and SMN-dependent pathway and further unveil the cellular pathway protecting against SMA.
The applicant has recently received the Innovationspreis NRW 2019, among others for the discovery of NCALD and CHP1 and their potential use for development of an SMA therapy.
Institute for Human Genetics / RG location - CMMC Building
Principal Investigator - C 16
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