Durable HTT silencing using non-evolved dCas9 epigenome editors in patient-derived cells

Jennifer J Waldo; Julian A N M Halmai; Ankita Singh; Casiana E Gonzalez; Yi-An Chen; Shaylyn A Carthen; Jan A Nolta; Kyle D Fink

doi:10.1016/j.omtn.2025.102561

Durable HTT silencing using non-evolved dCas9 epigenome editors in patient-derived cells

Mol Ther Nucleic Acids. 2025 May 14;36(2):102561. doi: 10.1016/j.omtn.2025.102561. eCollection 2025 Jun 10.

Authors

Jennifer J Waldo^{1

2

3

4

5

6}, Julian A N M Halmai^{1

2

3

4

5

6}, Ankita Singh^{1

2

3

4

5

6}, Casiana E Gonzalez^{1

2

3

4

5

6}, Yi-An Chen^{1

2

3

4

5

6}, Shaylyn A Carthen^{1

2

3

4

5

6}, Jan A Nolta^{3

4

5}, Kyle D Fink^{1

2

3

4

5

6}

Affiliations

¹ Ctr. for Interventional Genetics, University of California Davis Health, Sacramento, CA, USA.
² MIND Institute, University of California Davis Health, Sacramento, CA, USA.
³ Stem Cell Program, University of California Davis Health, Sacramento, CA, USA.
⁴ Gene Therapy Center, University of California Davis Health, Sacramento, CA, USA.
⁵ Institute for Regenerative Cures, University of California Davis Health, Sacramento, CA, USA.
⁶ Department of Neurology, University of California Davis Health, Sacramento, CA, USA.

Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat expansion in exon 1 of the huntingtin (HTT) gene. Nuclease-deficient Cas9 protein (dCas9) epigenetic editing for targeted gene regulation is a promising therapeutic approach for HD through downregulation of the causative gene, HTT. A screen of several dCas9 variants with expanded PAM recognition was fused to KRAB and DNMT3A/L to assess the ability to downregulate total HTT. Surprisingly, only S pdCas9 could significantly downregulate HTT, while expanded PAM recognition variants dxCas9 and dCas9-VQR were less efficient or unable to reduce HTT expression. Using our lead construct with S pdCas9, DNA methylation changes were assessed through reduced representation bisulfite sequencing, showing high on-target increases in DNA methylation and few off-targets. In addition, HTT silencing was mitotically stable for up to 6 weeks in a rapidly dividing cell line. Finally, significant downregulation of HTT was achieved in patient-derived neuronal stem cells, showing the efficacy of this system in a disease-relevant cell type. This approach represents a novel therapeutic pathway for the treatment of HD.

Keywords: CRISPR; DNA methylation; Huntington’s disease; MT: RNA/DNA Editing; dCas9; epigenetics.

Grants and funding

R01 NS102486/NS/NINDS NIH HHS/United States