Pharmacokinetics and pharmacodynamics of PTC518, an oral huntingtin lowering splicing modifier: A first-in-human study.

AIMS: PTC518 is an orally administered, centrally and peripherally distributed huntingtin (HTT) pre-mRNA splicing modifier being developed for the treatment of Huntington's disease (HD) for which there is a high unmet medical need as there are currently no approved disease-modifying treatments. This first-in-human study investigated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of PTC518 in healthy volunteers. METHODS: This phase 1, single-centre, randomized study in 77 healthy male and female volunteers evaluated the safety and tolerability and PK of PTC518 following single ascending doses and multiple ascending doses, PD as assessed by HTT mRNA and HTT protein levels after single and multiple doses, and food effects. RESULTS: PTC518 demonstrated a favourable safety profile. The majority of treatment-emergent adverse events were mild and transient. PTC518 Tmax was reached at 6-7 h and the terminal T1/2 was 54.0-75.3 h following a single oral dose. Exposure increased with dose though less than dose proportionally. The PTC518 concentrations in cerebrospinal fluid were approximately 2.6-fold higher than the unbound free-drug concentrations in plasma. A significant dose-dependent reduction of up to approximately 60% in HTT mRNA and a significant dose-dependent, time-dependent and sustained reduction in HTT protein levels of up to 35% were observed after PTC518 treatment. CONCLUSIONS: PTC518 was well tolerated, and proof of mechanism of this novel splicing modifier was demonstrated by the dose-dependent decrease in systemic HTT mRNA and HTT protein levels. Results from this first-in-human study support further studies in patients with HD and demonstrate the potential for PTC518 as a breakthrough treatment for HD.

Targeting strategies for modulating pre-mRNA splicing with small molecules: Recent advances.

The concept of using small molecules to therapeutically modulate pre-mRNA splicing was validated with the US Food and Drug Administration (FDA) approval of Evrysdi® (risdiplam) in 2020. Since then, efforts have continued unabated toward the discovery of new splicing-modulating drugs. However, the drug development world has evolved in the 10 years since risdiplam precursors were first identified in high-throughput screening (HTS). Now, new mechanistic insights into RNA-processing pathways and regulatory networks afford increasingly feasible targeted approaches. In this review, organized into classes of biological target, we compile and summarize small molecules discovered, devised, and developed since 2020 to alter pre-mRNA splicing.

Small molecule splicing modifiers with systemic HTT-lowering activity.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin (HTT) gene. Consequently, the mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin (HTT) protein levels alleviates motor and neuropathological abnormalities. Investigational drugs aim to reduce HTT levels by repressing HTT transcription, stability or translation. These drugs require invasive procedures to reach the central nervous system (CNS) and do not achieve broad CNS distribution. Here, we describe the identification of orally bioavailable small molecules with broad distribution throughout the CNS, which lower HTT expression consistently throughout the CNS and periphery through selective modulation of pre-messenger RNA splicing. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels.

Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.

Utility of survival motor neuron ELISA for spinal muscular atrophy clinical and preclinical analyses.

OBJECTIVES: Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA. METHODS: A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed. RESULTS: The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA. CONCLUSIONS: This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts.

Mining the GEMS--a novel platform technology targeting post-transcriptional control mechanisms.

The physiological levels of many clinically important proteins are regulated through cellular mechanisms that control the stability and translational efficiency of mRNA. These post-transcriptional processes, which play a critical role in the regulation of gene expression, depend on interactions of specific trans-acting factors with sequence elements located within the 5'- and 3'-untranslated regions (UTRs) of an mRNA. A technology platform called GEMS (Gene Expression Modulation by Small-molecules) exploits the interactions of UTR elements with the trans-acting factors, thereby specifically targeting mechanisms of post-transcriptional control. In this review we describe how this technology enables the identification of small-molecules that modulate the levels of proteins involved in disease pathogenesis.