Successfully tested in primates
A promising form of gene therapy called antisense oligonucleotides (ASOs) has been developed for a particular variant of SPG49 and tested in patient-derived neurons induced from skin cells.
A single dose was then tested in cynomolgus monkeys by direct injection into the cerebral spinal fluid with acceptable tolerability.
Later study of central nervous system tissues revealed the desired effect on the TECPR2 gene.
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.
Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models
Luis A. Williams,1,10 David J. Gerber,1,10 Amy Elder,1,10 Wei Chou Tseng,1,10 Valeriya Baru,1 Nathaniel Delaney-Busch,1 Christina Ambrosi,1 Gauri Mahimkar,1 Vaibhav Joshi,1 Himali Shah,1 Karthiayani Harikrishnan,1 Hansini Upadhyay,1 Sakthi H. Rajendran,1 Aishwarya Dhandapani,1 Joshua Meier,1 Steven J. Ryan,1 Caitlin Lewarch,1 Lauren Black,2 Julie Douville,2 Stefania Cinquino,2 Helen Legakis,2 Karsten Nalbach,3 Christian Behrends,3 Ai Sato,4 Lorenzo Galluzzi,4 Timothy W. Yu,5 Duncan Brown,1 Sudhir Agrawal,6,7 David Margulies,1 Alan Kopin,8,9,∗ and Graham T. Dempsey1,∗∗
1. Q-State Biosciences, 179 Sidney Street, Cambridge, MA 02139, USA
2. Charles River Laboratories, Montreal, QC, Canada
3. Munich Cluster for Systems Neurology, Ludwig-Maximilians-Universität München, Germany
4. Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA
5. Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
6. University of Massachusetts Medical School, Department of Medicine, Worcester, MA 01655, USA
7. Arnay Sciences LLC, Shrewsbury, MA 01545, USA
8. Tufts University School of Medicine, Boston, MA, USA
9. Luke Heller TECPR2 Foundation, Swampscott, MA, USA
*Corresponding author. Alan Kopin, Tufts University School of Medicine, Boston, MA, USA.
**Corresponding author. Graham T. Dempsey, Q-State Biosciences, 179 Sidney Street, Cambridge, MA 02139, USA.
These authors contributed equally.