The power of protective modifier NCALD to develop an efficient combinatorial therapy for spinal muscular atrophy

• Applicant: Professorin Dr. Brunhilde Wirth
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 398410809

• Final Report Year: 2023

Final Report Abstract

Neurocalcin delta (NCALD) is a brain-enriched neuronal calcium sensor and its reduction acts protective against spinal muscular atrophy (SMA). NCALD reduction ameliorates SMA in humans, as found in asymptomatic SMN1-deleted individuals and across genetically-modified SMA species such as mice, flies and C. elegans. In this project, we aimed to 1) investigate the effect of NCALD reduction in combination with a low-dose SMN-ASO in SMA mice to test the potency of Ncald-ASOs in SMA, 2) to find nontoxic and effective human NCALD-ASOs and to study their effects in human motor neuron differentiated from induced pluripotent stem cells (hiPSC), and 3) to uncover novel interacting partners of NCALD and potential pathways affected by Ncald knockdown or knockout in mice. In this project, we first performed a double-blind preclinical study of presymptomatic treatment of SMA and control mice with low-dose SMN-ASO (systemic injection) and Ncald-ASOs (CNS injection). In order to develop the best Ncald-ASOs, IONIS Pharmaceuticals (USA) designed and tested around 450-Ncald ASOs for our project, as part of a collaboration. The three best hits were further analyzed in neonatal mice in Cologne. The most efficient and non-toxic Ncald- ASO3 was then used in a double-blind preclinical study. Mice were subcutaneously injected with low dose SMN ASOs at PND1 and Ncald or control ASOs at PND2, and analyzed at PND21 and at 3-month-of-age. At PND21, we observed significant amelioration of histological and electrophysiological SMA hallmarks, but not at 3 months. This rather unexpected result was due to the reduced stability of Ncald-ASOs in vivo as compared to SMN-ASOs. Therefore, we decided to investigate the longer-term effects of Ncald-ASOs with additional i.c.v. bolus injection at PND28. Two weeks after injection of 500 µg Ncald-ASO in wild-type mice, NCALD was significantly reduced in brain and spinal cord and well tolerated. Therefore, we next performed a second double-blind preclinical study combining low-dose SMN-ASO (PND1) with 2x i.c.v. Ncald-ASO or CTRL-ASO (100 µg at PND2, 500 µg at PND28). Ncald-ASO re-injection significantly ameliorated electrophysiological defects and neuromuscular denervation at 2 months as compared to CTRL-ASO. Furthermore, we have developed and identified a nontoxic and highly efficient human NCALD- ASO that significantly reduced NCALD in motor neurons differentiated from hiPSCs. Like for the mice, IONIS Pharmaceuticals designed and tested a full battery of human NCALD-ASOs for this project. The three best hits were next investigated in motor neurons differentiated from hiPSC lines derived from two SMA and two control individuals in Cologne. Only treatment of motor neurons with NCALD-ASO69 was well tolerated, nontoxic and led to significant NCALD reduction, while the other two were toxic for MN development. Instead NCALD-ASO69 efficiently restored neuronal activity and growth cone maturation of SMA motor neurons to almost control levels. Lastly, in a functional study using co-immunoprecipitation and mass spectrometry from wildtype versus Ncaldko/ko spinal cord, we identified a novel interesting NCALD interaction partner, mitogen-activated protein kinase kinase kinase 10 (MAP3K10), and found that NCALD is essential in adult neurogenesis in mice, most likely via the MAP3K10-JNK pathway. Our studies funded by this DFG grant emphasize the additional protective role of NCALD-ASO treatment in mice and human iPSC-differentiated motor neurons. These data open the door for an initial pilot study in SMA patients.

Publications

• NCALD Antisense Oligonucleotide Therapy in Addition to Nusinersen further Ameliorates Spinal Muscular Atrophy in Mice. The American Journal of Human Genetics, 105(1), 221-230.
  Torres-Benito, Laura; Schneider, Svenja; Rombo, Roman; Ling, Karen K.; Grysko, Vanessa; Upadhyay, Aaradhita; Kononenko, Natalia L.; Rigo, Frank; Bennett, C. Frank & Wirth, Brunhilde
  
• Neurocalcin Delta Knockout Impairs Adult Neurogenesis Whereas Half Reduction Is Not Pathological. Frontiers in Molecular Neuroscience, 12 (2019, 2, 12).
  Upadhyay, Aaradhita; Hosseinibarkooie, Seyyedmohsen; Schneider, Svenja; Kaczmarek, Anna; Torres-Benito, Laura; Mendoza-Ferreira, Natalia; Overhoff, Melina; Rombo, Roman; Grysko, Vanessa; Kye, Min Jeong; Kononenko, Natalia L. & Wirth, Brunhilde
  
• Twenty-Five Years of Spinal Muscular Atrophy Research: From Phenotype to Genotype to Therapy, and What Comes Next. Annual Review of Genomics and Human Genetics, 21(1), 231-261.
  Wirth, Brunhilde; Karakaya, Mert; Kye, Min Jeong & Mendoza-Ferreira, Natalia
  
• Plastin 3 in health and disease: a matter of balance. Cellular and Molecular Life Sciences, 78(13), 5275-5301.
  Wolff, Lisa; Strathmann, Eike A.; Müller, Ilka; Mählich, Daniela; Veltman, Charlotte; Niehoff, Anja & Wirth, Brunhilde
  
• Spinal Muscular Atrophy: In the Challenge Lies a Solution. Trends in Neurosciences, 44(4), 306-322.
  Wirth, Brunhilde
  
• Combinatorial ASO-mediated therapy with low dose SMN and the protective modifier Chp1 is not sufficient to ameliorate SMA pathology hallmarks. Neurobiology of Disease, 171, 105795.
  Muinos-Bühl, A.; Rombo, R.; Janzen, E.; Ling, K.K.; Hupperich, K.; Rigo, F.; Bennett, C.F. & Wirth, B.
  
• Long-Term SMN- and Ncald-ASO Combinatorial Therapy in SMA Mice and NCALD-ASO Treatment in hiPSC-Derived Motor Neurons Show Protective Effects. International Journal of Molecular Sciences, 24(4), 4198.
  Muiños-Bühl, Anixa; Rombo, Roman; Ling, Karen K.; Zilio, Eleonora; Rigo, Frank; Bennett, C. Frank & Wirth, Brunhilde