Marla Weetall

PTC124 targets genetic disorders caused by nonsense mutations

Nonsense mutations promote premature translational termination and cause anywhere from 5–70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124—a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2–8u2009weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy

Drugs that provide the splice of life Motor neurons relay signals from the nervous system to muscle fibers. In patients with spinal muscular atrophy, a protein required for the survival of these neurons is deficient or missing altogether, so the neurons gradually die and the patients muscles waste away. The disease is currently untreatable. Naryshkin et al. discovered small-molecule drugs that cause cells to produce the missing protein by altering how a specific mRNA is put together, or “spliced” (see the Perspective by Vigevani and Valcárcel). When the researchers used the drugs to treat diseased mice, the mice showed marked improvement in muscle mass, motor function, and survival. Science, this issue p. 688; see also p. 624 Small molecules that alter splicing of a specific messenger RNA have beneficial effects in a model of a motor neuron disease. [Also see Perspective by Vigevani and Valcárcel] 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.

BMI1 as a novel target for drug discovery in cancer

Growing evidence has demonstrated that clonogenic cancer stem (initiating) cells are responsible for tumor regrowth and disease relapse. Bmi‐1 plays a critical role in the self‐renewal of adult stem cells. The Bmi‐1 protein is elevated in many types of cancers, and experimental reduction of Bmi‐1 protein levels by small interfering RNA (siRNA) causes apoptosis and/or senescence in tumor cells in vitro and increases susceptibility to cytotoxic agents. The Bmi‐1 protein has no known enzymatic activity, but serves as the key regulatory component of the PRC1 complex (polycomb repressive complex‐1). This complex influences chromatin structure and regulates transcriptional activity of a number of important loci including the Ink4a locus which encodes the tumor suppressor proteins p16Ink4a and p14Arf. In this prospective study, we will discuss the implication of BMI1 in cancers, the biology of BMI1, and the regulatory control of BMI1 expression. The target validation and the future prospects of targeting BMI1 in cancer therapy are also discussed. J. Cell. Biochem. 112: 2729–2741, 2011.

Functional rescue of REP1 following treatment with PTC124 and novel derivative PTC-414 in human choroideremia fibroblasts and the nonsense-mediated zebrafish model

Choroideremia (CHM) is an X-linked chorioretinal dystrophy that is caused by mutations within a single gene, CHM Currently no effective treatment exists for these patients. Since over 30% of patients harbour nonsense mutations in CHM, nonsense suppression therapy using translational readthrough inducing drugs may provide functional rescue of REP1, thus attenuating progressive sight loss. Here, we employed two CHM model systems to systematically test the efficacy and safety of ataluren (PTC124) and its novel analog PTC-414: (1) the chmru848 zebrafish, the only nonsense mutation animal model of CHM harbouring a TAA nonsense mutation, and (2) a primary human fibroblast cell line from a CHM patient harbouring a TAG nonsense mutation. PTC124 or PTC-414 treatment of chmru848 embryos led to a ∼2.0-fold increase in survival, prevented the onset of retinal degeneration with reduced oxidative stress and apoptosis, increased rep1 protein by 23.1% (PTC124) and 17.2% (PTC-414) and restored biochemical function as confirmed through in vitro prenylation assays (98u2009±u20092% [PTC124] and 68u2009±u20095% [PTC-414]). In CHMY42X/y fibroblasts, there was a recovery of prenylation activity following treatment with either PTC124 (42u2009±u20095%) or PTC-414 (36u2009±u200911%), although an increase in REP1 protein was not detected in these cells, in contrast to the zebrafish model. This comprehensive study on the use of PTC124 and PTC-414 as successful nonsense suppression agents for the treatment of CHM highlights the translational potential of these drugs for inherited retinal disease.

Pharmacologically induced mouse model of adult spinal muscular atrophy to evaluate effectiveness of therapeutics after disease onset

Spinal muscular atrophy (SMA) is a genetic disease characterized by atrophy of muscle and loss of spinal motor neurons. SMA is caused by deletion or mutation of the survival motor neuron 1 (SMN1) gene, and the nearly identical SMN2 gene fails to generate adequate levels of functional SMN protein due to a splicing defect. Currently, several therapeutics targeted to increase SMN protein are in clinical trials. An outstanding issue in the field is whether initiating treatment in symptomatic older patients would confer a therapeutic benefit, an important consideration as the majority of patients with milder forms of SMA are diagnosed at an older age. An SMA mouse model that recapitulates the disease phenotype observed in adolescent and adult SMA patients is needed to address this important question. We demonstrate here that Δ7 mice, a model of severe SMA, treated with a suboptimal dose of an SMN2 splicing modifier show increased SMN protein, survive into adulthood and display SMA disease-relevant pathologies. Increasing the dose of the splicing modifier after the disease symptoms are apparent further mitigates SMA histopathological features in suboptimally dosed adult Δ7 mice. In addition, inhibiting myostatin using intramuscular injection of AAV1-follistatin ameliorates muscle atrophy in suboptimally dosed Δ7 mice. Taken together, we have developed a new murine model of symptomatic SMA in adolescents and adult mice that is induced pharmacologically from a more severe model and demonstrated efficacy of both SMN2 splicing modifiers and a myostatin inhibitor in mice at later disease stages.

SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy.

Pharmacokinetics, pharmacodynamics, and efficacy of a small-molecule SMN2 splicing modifier in mouse models of spinal muscular atrophy

Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a ∼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment.

The nucleoside analog clitocine is a potent and efficacious readthrough agent

Nonsense mutations resulting in a premature stop codon in an open reading frame occur in critical tumor suppressor genes in a large number of the most common forms of cancers and are known to cause or contribute to the progression of disease. Low molecular weight compounds that induce readthrough of nonsense mutations offer a new means of treating patients with genetic disorders or cancers resulting from nonsense mutations. We have identified the nucleoside analog clitocine as a potent and efficacious suppressor of nonsense mutations. We determined that incorporation of clitocine into RNA during transcription is a prerequisite for its readthrough activity; the presence of clitocine in the third position of a premature stop codon directly induces readthrough. We demonstrate that clitocine can induce the production of p53 protein in cells harboring p53 nonsense-mutated alleles. In these cells, clitocine restored production of full-length and functional p53 as evidenced by induced transcriptional activation of downstream p53 target genes, progression of cells into apoptosis, and impeded growth of nonsense-containing human ovarian cancer tumors in xenograft tumor models. Thus, clitocine induces readthrough of nonsense mutations by a previously undescribed mechanism and represents a novel therapeutic modality to treat cancers and genetic diseases caused by nonsense mutations.

Discovery of Novel Small Molecule Inhibitors of VEGF Expression in Tumor Cells Using a Cell-Based High Throughput Screening Platform

Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal “homeostatic” state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5’ and 3’ untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies.

PTC124 for cystic fibrosis – Authors' reply

1426 www.thelancet.com Vol 373 April 25, 2009 years is indiff erent. Diff erent decisionmakers might permissibly weigh these principles diff erently. The complete lives system does not solve all diffi cult cases. No theory can do that. But it does provide a clear, systematic framework for analysing the cases and indicating what tradeoff s are being made. Gandjour grants that the rule of rescue only seems warranted if “it actually saves lives”. The rule of rescue is a means to achieving a morally important end. If another moral principle, such as youngest-fi rst or lottery allocation, is better at saving lives, we should use that principle instead. The rule of rescue might be “inborn and intuitive.” However, having an uncritical attitude towards our intuitive impulses is a failure of the moral imagination. In addition to pity, we have many violent and unjust inborn and intuitive responses that need to be suppressed rather than indulged. Over history, public executions and displays of violence have viscerally delighted people. Accepting the inborn and intuitive might be what often allows moral injustice to go unchecked.