Molecular Therapy: Nucleic Acids (Sep 2022)

Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models

  • Luis A. Williams,
  • David J. Gerber,
  • Amy Elder,
  • Wei Chou Tseng,
  • Valeriya Baru,
  • Nathaniel Delaney-Busch,
  • Christina Ambrosi,
  • Gauri Mahimkar,
  • Vaibhav Joshi,
  • Himali Shah,
  • Karthiayani Harikrishnan,
  • Hansini Upadhyay,
  • Sakthi H. Rajendran,
  • Aishwarya Dhandapani,
  • Joshua Meier,
  • Steven J. Ryan,
  • Caitlin Lewarch,
  • Lauren Black,
  • Julie Douville,
  • Stefania Cinquino,
  • Helen Legakis,
  • Karsten Nalbach,
  • Christian Behrends,
  • Ai Sato,
  • Lorenzo Galluzzi,
  • Timothy W. Yu,
  • Duncan Brown,
  • Sudhir Agrawal,
  • David Margulies,
  • Alan Kopin,
  • Graham T. Dempsey

Journal volume & issue
Vol. 29
pp. 189 – 203

Abstract

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Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with ∼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.

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