An alternative splicing modulator decreases mutant HTT and improves the molecular fingerprint in Huntington’s disease patient neurons

Abstract

Huntington’s disease (HD) is a neurodegenerative disorder caused by poly-Q expansion in the Huntingtin (HTT) protein. Here, we delineate elevated mutant HTT (mHTT) levels in patient-derived cells including fibroblasts and iPSC derived cortical neurons using mesoscale discovery (MSD) HTT assays. HD patients’ fibroblasts and cortical neurons recapitulate aberrant alternative splicing as a molecular fingerprint of HD. Branaplam is a splicing modulator currently tested in a phase II study in HD (NCT05111249). The drug lowers total HTT (tHTT) and mHTT levels in fibroblasts, iPSC, cortical progenitors, and neurons in a dose dependent manner at an IC₅₀ consistently below 10 nM without inducing cellular toxicity. Branaplam promotes inclusion of non-annotated novel exons. Among these Branaplam-induced exons, there is a 115 bp frameshift-inducing exon in the HTT transcript. This exon is observed upon Branaplam treatment in Ctrl and HD patients leading to a profound reduction of HTT RNA and protein levels. Importantly, Branaplam ameliorates aberrant alternative splicing in HD patients’ fibroblasts and cortical neurons. These findings highlight the applicability of splicing modulators in the treatment of CAG repeat disorders and decipher their molecular effects associated with the pharmacokinetic and -dynamic properties in patient-derived cellular models.

Introduction

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by CAG-repeat expansion in the coding region of the Huntingtin (HTT) transcript, leading to an elongated polyglutamine (poly-Q) stretch in HTT¹. While individuals with 40 and above CAG-repeats in one allele will develop HD, a repeat length below 36 is non-pathogenic. Intermediate repeat lengths in the range between 36 and 39 may cause HD with incomplete penetrance². The elongated poly-Q, mutant HTT (mHTT) is suspected to exhibit a toxic gain of function resulting in neuronal toxicity³.

Clinically, HD is characterized by the triad of motor dysfunction, psychiatric symptoms, and cognitive deficits. Motor symptoms, most prominently chorea, are related to the degeneration of striatal medium spiny neurons² and subsequently cortical projection neurons. Interestingly, cortico-striatal projections are affected years prior to disease onset⁴. Specifically, degeneration of pyramidal neurons in the primary motor cortex and anterior cingulate cortex lead to more pronounced motor and mood impairments, respectively^5,6.

So far, there is no causal therapy to improve or even halt the disease course of HD. However, a number of recent clinical trials focus on small compounds lowering HTT levels⁷. One of the latest clinical trials investigates the effectiveness of the alternative splicing (AS) modulator Branaplam (previously called LMI070, NVS-SM1) in HD (NCT05111249). Branaplam was initially designed for spinal muscular atrophy (SMA) and promotes inclusion of exon 7 in the SMN2 transcript via stabilization of the pre-mRNA – U1 snRNP complex⁸.

Here, we describe Branaplam’s mechanism of action and effects in HD. We develop a HD patient-derived cellular platform to investigate mHTT levels using a validated HTT assay. We demonstrate that Branaplam is able to reduce mHTT levels. Furthermore, Branaplam restores mHTT-induced aberrant splicing, an important molecular feature of HD, in transcripts that are not directly targeted by Branaplam. We evaluate the precise pharmacokinetic and -dynamic properties of Branaplam in primary fibroblasts and iPSC-derived cortical neurons of Ctrl and HD patients. Lastly, we explore the small molecule’s potential to revert molecular phenotypes of HD patients’ neurons.

Results

mHTT is increased in cellular model of HD patients

We chose to use various patient-derived non-neuronal and neuronal cells to model HD in vitro (Fig. 1a). Besides four Ctrls, we recruited four HD patients originating from three distinct families (Table 1 and Supplementary Fig. 1a). The CAG repeats on the affected allele ranged from 39 to 57 (Table 1). Clinically, all patients presented motor symptoms. Hence, we reprogrammed fibroblasts into iPSC and subsequently differentiated them into cortical neurons using a previously published dual-SMAD-inhibition-based protocol⁹. At day 25 of differentiation, NESTIN⁺/PAX6⁺ cortical progenitors were observed (Fig. 1b and Supplementary Fig. 1b, c), and from day 35 on deep layer cortical neurons positive for CTIP2 were apparent (Fig. 1c, d and Supplementary Fig. 1d, e). No differences in differentiation potential between Ctrl and HD were detected (Fig. 1b, c)….