International Journal of Molecular Sciences Review Huntingtin Lowering Strategies Franz Marxreiter 1,2,*, Judith Stemick 1 and Zacharias Kohl 3 1 Huntington’s Disease Outpatient Clinic, Department of Molecular Neurology, University Hospital Erlangen, Schwabachanlage 6, 91054 Erlangen, Germany; [email protected] 2 Center for Rare Movement Disorders, Department of Molecular Neurology, University Hospital Erlangen, Schwabachanlage 6, 91054 Erlangen, Germany 3 Department of Neurology, University of Regensburg, Universitätsstraße 84, 93053 Regensburg, Germany; [email protected] * Correspondence: [email protected] Received: 26 February 2020; Accepted: 17 March 2020; Published: 20 March 2020 Abstract: Trials using antisense oligonucleotide technology to lower Huntingtin levels in Huntington’s disease (HD) are currently ongoing. This progress, taking place only 27 years after the identification of the Huntingtin gene (HTT) in 1993 reflects the enormous development in genetic engineering in the last decades. It is also the result of passionate basic scientific work and large worldwide registry studies that have advanced the understanding of HD. Increased knowledge of the pathophysiology of this autosomal dominantly inherited CAG-repeat expansion mediated neurodegenerative disease has led to the development of several putative treatment strategies, currently under investigation. These strategies span the whole spectrum of potential targets from genome editing via RNA interference to promoting protein degradation. Yet, recent studies revealed the importance of huntingtin RNA in the pathogenesis of the disease. Therefore, huntingtin-lowering by means of RNA interference appears to be a particular promising strategy. As a matter of fact, these approaches have entered, or are on the verge of entering, the clinical trial period. Here, we provide an overview of huntingtin-lowering approaches via DNA or RNA interference in present clinical trials as well as strategies subject to upcoming therapeutic options. We furthermore discuss putative implications for future treatment of HD patients. Keywords: Huntington’s Disease; huntingtin-lowering; antisense oligonucleotides; RNA interference; disease modification; Chorea; HD 1. Introduction 1.1. Huntington’s Disease Huntington’s disease (HD) is clinically characterized by a triad of movement disorders, psychiatric symptoms and cognitive deficits. While the term Huntington’s chorea focuses solely on the hyperkinetic movements, chorea is not the only movement disorder in HD. Dystonia and bradykinesia are common as well [1]. They may even be the sole movement abnormality, especially in juvenile HD. The disease manifests as a movement disorder in about 60% of cases, cognitive deficits dominate the initial clinical picture in 14% and psychiatric abnormalities are the initial presentation in about 20% of cases. Initial cognitive deficits consist of executive dysfunction, concentration and attention deficits, and a disturbed ability to plan. Psychiatric symptoms include irritability, aggressive behavior and disturbed impulse control or apathy [2]. However, the diagnosis of clinically manifest HD is currently exclusively based on the presence of motor symptoms [3]. Current pharmacotherapy in HD is limited to symptomatic treatment of movement disorders and psychiatric symptoms. The only approved drug for the therapy of hyperkinesia and dystonia in HD is tetrabenazine [4]. Despite numerous clinical Int. J. Mol. Sci. 2020, 21, 2146; doi:10.3390/ijms21062146 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2146 2 of 13 studies on disease-modifying strategies (see e.g., homepage of the Huntington Study Group), thus far, no therapy exists that is able to slow down, stop or even reverse the course of the disease. 1.2. Genetics and Pathophyiology of Huntington’s Disease HD is a rare autosomal dominantly inherited neurodegenerative disease with worldwide prevalence of 1–10/100,000 [5]. In Europe, a prevalence of 3/100,000 is assumed, the highest prevalence occurs in Sicily with 9.8/100,000 affected islanders [6]. However, current studies show that the genotype frequency with intermediate (36–39) and pathological (>39) expansion in the huntigtin gene (HTT) may be as high as 139/100,000 [7]. Genetically, HD belongs to the group of trinucleotide repeat expansion disorders. While individuals with a CAG repeat length of less than 35 located on exon 1 of HTT remain asymptomatic, alleles with 40 or more repeats show full penetrance. Intermediate alleles (36–39 CAG repetitions) bear an increased risk to develop HD. There is an inverse correlation between the age of onset, and the length of the CAG repeat expansion, whereby alleles with more than 70 repetitions always cause a manifestation in adolescence. The mean age of onset of this progressive and life limiting disease is 40 years, the average disease duration is 20 years (overview in [8]). Even in early stages, the quality of life and the social environment of those affected is severely impaired. Transcription of pathologically prolonged HTT into prolonged huntingtin (htt) pre-mRNA, its processing to mRNA, and translation into a prolonged protein lead to endoplasmatic reticulum stress (reviewed in [9]), misfolding and aggregation (Figure1 ), particularly in neurons. Additionally, several toxic short protein species have been described. Some of these species are the result of proteolytic cleavage [10]. The repeat length of the second allele does not influence disease onset or progression [11]. The CAG repeat expansion alone accounts for 50%–70% of the variance in disease onset. Interestingly, the number of uninterrupted CAG repeats, distinct from the length of the polyglutamine segment of the htt protein, accounts for the age of onset of the disease [12]. Several genetic modifiers, most of which represent DNA repair genes, influence the onset of the disease as well [13]. An additional contributor to pathogenesis may be epigenetic modification; in particular, histone deacetylation (HDAC) has repeatedly been shown to negatively influence disease onset. Thus, HDAC inhibition is a promising disease modifying strategy [14]. Splice variants and Repeat Associated Non-AUG (RAN) translation can also lead to neurotoxic isoforms of htt [15]. In addition, there is increasing evidence that a pathologically prolonged htt mRNA leads to defective binding of RNA binding proteins, further contributing to pathogenesis (overview in [15]) (Figure1B). Prolonged htt protein acts primarily through a toxic gain-of-function, interfering with autophagy, vesicle transport, neurotransmitter release and mitochondrial function (Figure1C). In summary, this results in the degeneration of neurons, predominantly striatal and cortical GABAergic neurons (reviewed in [8]). Furthermore, inflammatory processes also occur, which trigger the progression of the disease (overview in [16]). Thus, from a pathophysiological standpoint, disease-modifying therapies targeting the pathological processing of htt mRNA, or upstream, appear to be the most promising. 1.3. The Scientific Basis for Gene Expression Modification in HD; From the Discovery of the HD Gene to Clinical Trials In 1983, the Venezuela Project, a field study at Lake Maracaibo in Venezuela, carried out by the World Hereditary Disease Foundation, succeeded in mapping HTT on the short arm of chromosome 4 [17]. Following the first discovery of a trinucleotide expansion disorder (fragile X syndrome) in 1991 [18], a CAG repeat expansion in HTT was subsequently identified in 1993 as the disease-causing alteration [19], and genetic testing became possible. Already the first murine model of HD in 1996 [20] was able to recapitulate essential aspects of the disease. In 2000, a tetracycline-regulated conditional mouse model in which the expression of mutated HTT was suppressed by doxycycline treatment, showed reversibility of brain pathology and behavioral deficits by transgene suppression [21]. Positive effects on RNA interference were later observed in a murine model [22]. Kordasiewicz et al. [23] Int. J. Mol. Sci. 2020, 21, 2146 3 of 13 succeeded in lowering htt in three murine models of HD by using an antisense oligonucleotide (ASO) in 2012, also achieving an improvement of motor symptoms. Additionally, the authors showed that ASO infusion could reduce htt deposits in brains of a primate model. Thus, this study provided decisive preclinical evidence for the concept of “huntingtin holidays” as the basic principle of a disease-modifying strategy for HD patients [24]. Sensitive and standardized immunoassays, which allow the quantification of mutated htt protein in cerebrospinal fluid [25,26], have become available as biomarkers [27]. 2. Genetic Engineering Approaches for Disease Modification in HD Disease modification by lowering htt levels via interfering with transcription has been investigated at different levels. Antisense oligonucleotides (ASOs) [23], single-stranded RNA molecules (ssRNAs), small interfering RNAs (siRNAs) [28] and short hairpin RNA (shRNAs) [22] or artificial micro RNA (miRNAs) [29] were used for this purpose. All of these approaches interfere on RNA level and are able to suppress htt levels in vitro and in animal models. With respect to clinical testing however, the utilization of ASOs is the most advanced strategy (Figure2). By using ASOs, gene expression is only downregulated transiently, and repetitive application is necessary to maintain constant lowering of htt levels. Therefore, these approaches are not called ”gene therapy,”