Personalized Splice-Modifying ASOs Correct Pathogenic AP4M1 Splicing and Improve AP-4 Functionality in Cultured SPG50 Patient Cells

Topic:

Translational Research

Poster Number: 78 S

Author(s):

Jack Howell, BS, National Institute for Neurological Disease and Stroke, Lindsey Trank, BS, Ohio University Heritage College of Osteopathic Medicine, Raffaella De Pace, PhD, Institute of Child Health and Human Development, National Institutes of Health, Véronique Bolduc, PhD, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Ying Hu, MS, Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Health, Sandra Donkervoort, MS, Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Healt, Rotem Orbach, MD, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Darius Ebrahimi-Fakhari, MD, PhD, Boston Children’s Hospital, Juan Bonifacino, PhD, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Carsten G. Bönnemann, MD, habil., Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, National Institutes of Health

Hereditary Spastic Paraplegias (HSPs) are the leading cause of inherited spasticity and related disability, marked by progressive lower limb weakness and spasticity with variable rates of progression. One subtype of interest, SPG50, causes a prototypical HSP phenotype combined with intellectual disability and neuroanatomical changes. SPG50 is caused by biallelic pathogenic variants in the μ4 subunit (AP4M1) of the heterotetrameric adaptor protein complex 4 (AP-4). AP-4 mediates export of several cargos, including the autophagy protein ATG9A, from the trans-Golgi network.

Given its clear genetic etiology and significant disease burden, SPG50 is a promising candidate for gene replacement therapies, with gene transfer trials already underway. However, we recently identified one patient who may also benefit from a complementary splice-modifying therapeutic. This patient displayed the progressive spasticity and biallelic variants in AP4M1 characteristic of SPG50. Their paternally inherited allele contains a terminating frameshift mutation (c.1129del; p.Leu377PhefsTer67) and their maternally inherited allele an intronic duplication (c.974+115dup) that creates a donor splice site, extending exon 12 by 112 nucleotides and introducing a premature stop codon. Because haploinsufficiency of AP4M1 is asymptomatic, reverting splicing to the canonical donor site with an antisense oligonucleotide (ASO) in their maternal allele could meaningfully improve patient symptoms.

To explore this approach, we designed two mutation-targeted phosphorothioate 2’MOE ASOs. In cultured patient fibroblasts, one ASO reduced aberrant splicing, as assayed with RT-dPCR, and improved AP-4 functionality as evidenced by corrected AP-4 cargo protein localization. Ongoing studies are evaluating the impact of ASOs at improving these same markers in iPSC-derived neuronal cells. Overall, this work demonstrates the promise of n-of-1 splice-correcting therapies for ultrarare neurological disorders and their complementarity to viral gene transfer approaches.