A pathogenic proteolysis–resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function
Hyeongju Kim, Sophie Lenoir, Angela Helfricht, Taeyang Jung, Zhana K. Karneva, Yejin Lee, Wouter Beumer, Geert B. van der Horst, Herma Anthonijsz, Levi C.M. Buil, Frits van der Ham, Gerard J. Platenburg, Pasi Purhonen, Hans Hebert, Sandrine Humbert, Frédéric Saudou, Pontus Klein, Ji-Joon Song
Hyeongju Kim, Sophie Lenoir, Angela Helfricht, Taeyang Jung, Zhana K. Karneva, Yejin Lee, Wouter Beumer, Geert B. van der Horst, Herma Anthonijsz, Levi C.M. Buil, Frits van der Ham, Gerard J. Platenburg, Pasi Purhonen, Hans Hebert, Sandrine Humbert, Frédéric Saudou, Pontus Klein, Ji-Joon Song
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Research Article Neuroscience

A pathogenic proteolysis–resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function

Abstract

Huntington’s disease (HD) is a late-onset neurological disorder for which therapeutics are not available. Its key pathological mechanism involves the proteolysis of polyglutamine-expanded (polyQ-expanded) mutant huntingtin (mHTT), which generates N-terminal fragments containing polyQ, a key contributor to HD pathogenesis. Interestingly, a naturally occurring spliced form of HTT mRNA with truncated exon 12 encodes an HTT (HTTΔ12) with a deletion near the caspase-6 cleavage site. In this study, we used a multidisciplinary approach to characterize the therapeutic potential of targeting HTT exon 12. We show that HTTΔ12 was resistant to caspase-6 cleavage in both cell-free and tissue lysate assays. However, HTTΔ12 retained overall biochemical and structural properties similar to those of wt-HTT. We generated mice in which HTT exon 12 was truncated and found that the canonical exon 12 was dispensable for the main physiological functions of HTT, including embryonic development and intracellular trafficking. Finally, we pharmacologically induced HTTΔ12 using the antisense oligonucleotide (ASO) QRX-704. QRX-704 showed predictable pharmacology and efficient biodistribution. In addition, it was stable for several months and inhibited pathogenic proteolysis. Furthermore, QRX-704 treatments resulted in a reduction of HTT aggregation and an increase in dendritic spine count. Thus, ASO-induced HTT exon 12 splice switching from HTT may provide an alternative therapeutic strategy for HD.

Authors

Hyeongju Kim, Sophie Lenoir, Angela Helfricht, Taeyang Jung, Zhana K. Karneva, Yejin Lee, Wouter Beumer, Geert B. van der Horst, Herma Anthonijsz, Levi C.M. Buil, Frits van der Ham, Gerard J. Platenburg, Pasi Purhonen, Hans Hebert, Sandrine Humbert, Frédéric Saudou, Pontus Klein, Ji-Joon Song

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Figure 8

QRX-704 treatment of YAC128 mice reduces N-terminal cleavage fragments, alleviates HTT aggregation, and increases the number of dendritic spines.

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QRX-704 treatment of YAC128 mice reduces N-terminal cleavage fragments, ...
(A) Schematic of 10-month in vivo study performed in YAC128 mice dosed twice with 250 μg QRX-704 or aCSF (vehicle). (B) Western blot of different HTT species (HTTΔ12 or canonical isoform, full-length, C-terminal, and N-terminal fragments) of cortex from aCSF- or QRX-704–treated YAC128 mice with Abs as depicted in figure. HTTΔ12 mRNA (% of total) in the same tissue sample for each animal is depicted above the top panel. (C) N-terminal fragments’ relative quantification (1C2 Ab) normalized by vinculin signal in animals treated with aCSF (gray, n = 7) or QRX-704 (blue), separated into animals with HTTΔ12 mRNA less than 5% (open circles, n = 5) or greater than 5% (filled circles, n = 5), with mean ± SEM. (D) C-terminal fragments (D7F7 Ab) normalized by vinculin, analyzed as in B. (E) Full-length human HTT (D7F7 Ab) normalized by vinculin, analyzed as in B. (F) Immunostaining of sagittal brain sections of cortex and striatum from the right hemisphere of YAC128 mice from A. Representative images for HTT aggregation visualized by EM48 staining (red) and DAPI (blue) staining are shown for the indicated groups (n = 5 × 5 images recorded per location): untreated (dark gray), aCSF (light gray) and QRX-704 with HTTΔ12 greater than 5% (blue). (G) Quantification of the number of EM48-positive cells in recorded images from cortex and striatum. (H) The same as F, but immunostained for spinophilin indicating the number of dendritic spines. Representative images show spinophilin puncta, which were counted in an automated fashion. (I) Quantification of the number of spinophilin-positive puncta using ImageJ (NIH) threshold and counting tools on the recorded images from cortex and striatum. *P < 0.05, **P < 0.01, ****P < 0.0001. Statistics: 1-way ANOVA followed by Tukey’s multiple comparison test.