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Haplotyping individual full-length transcripts can be important in diagnosis and treatment of certain genetic diseases. One set of diseases, repeat expansions of simple tandem repeat sequences are the cause of over 40 neurological disorders. In many of these conditions, expanding a polymorphic repeat beyond a given threshold has been strongly associated with disease onset and severity. Given that most repeat expansions are inherited in an autosomal dominant pattern, repeat expansion disorders are typically characterized by a heterozygous expansion locus associated with a single haplotype. Precision genetic medicines can be used to selectively target expansion-containing sequences in a haplotype-specific manner.However, repeat expansion lengths often exceed the capacity of next-generation sequencing (NGS) reads. Therefore, the accurate length and haplotype determination of repeat expansions requires special considerations and requires the development of custom methods. Here we highlight a method for targeted haplotype phasing of the HTT gene, which can be adopted for use with other full-length transcripts and in other repeat expansion disorders.

As oligonucleotides (ONs) and similar nucleic acid therapeutic modalities enter development pipelines, there is continual need to develop bioanalytical methodologies addressing unique challenges they pose. Novel ONs back bone chemistries, especially those enabling stereochemical control, and base modifications are being exploited to improve pharmacological properties, potency, and increase half-lives. These changes have strained established methods, oftentimes precluding development of assays sensitive and specific enough to meet the needs of preclinical programs. For stereopure ONs representing a single molecular species, nontrivial presence of chain-shortened metabolites in biological samples necessitate assays with high specificity. To meet these needs, this report presents a toolbox of novel techniques, easy to implement for existing hybridization-ligation enzyme-linked immunosorbent assay formats, which address this challenge and yield significant sensitivity and specificity enhancements. Ligation efficiency was improved up to 61-fold through addition of polyethylene glycol, betaine, or dimethylsulfoxide, mitigating major differences among sequence-matched ONs of varying stereopurity, enabling sensitivities below 0.100 ng/mL for quantitation. These improvements enabled further refinement of capture probe designs engendering sufficient specificity to discriminate N-1 chain-shortened metabolites at both the 5' and 3' end of the ONs. These generalizable methods advance the performance of mainstay bioanalytical assays, facilitating research and development of innovative ONs therapeutics.

Novel treatments for Huntington’s disease (HD), a progressive neurodegenerative disorder, include selective targeting of the mutant allele of the huntingtin gene (mHTT) carrying the abnormally expanded disease-causing cytosine-adenine-guanine (CAG) repeat. WVE-120101 and WVE-120102 are investigational stereopure antisense oligonucleotides that enable selective suppression of mHTT by targeting single-nucleotide polymorphisms (SNPs) that are in haplotype phase with the CAG repeat expansion. Recently developed long-read sequencing technologies can capture CAG expansions and distant SNPs of interest and potentially facilitate haplotype-based identification of patients for clinical trials of oligonucleotide therapies. However, improved methods are needed to phase SNPs with CAG repeat expansions directly and reliably without need for familial genotype/haplotype data. Our haplotype phasing method uses single-molecule real-time sequencing and a custom algorithm to determine with confidence bases at SNPs on mutant alleles, even without familial data. Herein, we summarize this methodology and validate the approach using patient-derived samples with known phasing results. Comparison of experimentally measured CAG repeat lengths, heterozygosity, and phasing with previously determined results showed improved performance. Our methodology enables the haplotype phasing of SNPs of interest and the disease-causing, expanded CAG repeat of the huntingtin gene, enabling accurate identification of patients with HD eligible for allele-selective clinical studies., Graphical Abstract, Svrzikapa et al. describe a method using single-molecule real-time long-read sequencing and a custom algorithm to determine bases at SNPs on mutant alleles. Accurate haplotype phasing of SNPs in relationship to the expanded CAG repeat of the huntingtin gene enables identification of patients with Huntington’s disease eligible for allele-selective clinical studies.

A GGGGCC repeat expansion in theWe used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by theWe show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40

A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. We utilised the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the repeat expansion in CSF of people with C9orf72-associated FTD/ALS. We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intra- and inter-plate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) DPRs in the CSF of samples collected through the Genetic FTD Initiative. We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had 8-fold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.

Background Huntington’s disease is caused by the expansion of CAG-repeats (≥36 repeats) in at least one copy of HTT gene that leads to the expression of mutant HTT (mHTT) protein. The unaffected copy of HTT gene encodes wild-type HTT (wtHTT) protein, which supports many processes important for the health and function of the CNS. Aims Wave has developed WVE-003, an investigational stereopure oligonucleotide designed to selectively reduce the expression of mHTT mRNA while preserving wtHTT mRNA by targeting a single nucleotide polymorphism, SNP3, in the mHTT mRNA. WVE-003 contains Wave’s new PN chemistry, which has been shown to improve the pharmacological profile of oligonucleotides in preclinical studies. We are evaluating WVE-003 in an adaptive, multicenter, randomized, double-blind, placebo-controlled, phase 1b/2a clinical trial called SELECT-HD. The study is planned to enroll approximately 36 participants who have SNP3 only on the mHTT allele. We will assess the safety and tolerability of single- and multiple-ascending doses of WVE-003 administered intrathecally (IT) by lumbar puncture. Secondary objectives include studying WVE-003 pharmacokinetics (PK) in plasma and cerebrospinal fluid (CSF). We will also evaluate biomarkers for neurodegeneration, such as neurofilament light chain (NfL) in the CSF, as well as mutant and wild-type HTT proteins in CSF, biomarkers of pharmacodynamic effect and selectivity, respectively. The trial is designed to be adaptive, so that PK, safety and tolerability results from each cohort will inform the dose and dosing frequency for subsequent cohorts in the single- and multiple-ascending dose phases of the trial. Conclusions This first-in-human study will provide proof of concept for PD effects, as well as PK, safety and tolerability of WVE-003 in early manifest Huntington’s disease.

A Synechococcus sp. strain PCC 7002 Fe bioreporter was constructed containing the isiAB promoter fused to the Vibrio harveyi luxAB genes. Bioreporter luminescence was characterized with respect to the free ferric ion concentration in trace metal-buffered synthetic medium. The applicability of the Fe bioreporter to assess Fe availability in the natural environment was tested by using samples collected from the Baltic Sea and from the high-nutrient, low-chlorophyll subarctic Pacific Ocean. Parallel assessment of dissolved Fe and bioreporter response confirmed that direct chemical measurements of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton.

Background S-nitrosoglutathione (GSNO) serves as a reservoir for nitric oxide (NO) and thus is a key homeostatic regulator of airway smooth muscle tone and inflammation. Decreased levels of GSNO in the lungs of asthmatics have been attributed to increased GSNO catabolism via GSNO reductase (GSNOR) leading to loss of GSNO- and NO- mediated bronchodilatory and anti-inflammatory actions. GSNOR inhibition with the novel small molecule, N6022, was explored as a therapeutic approach in an experimental model of asthma. Methods Female BALB/c mice were sensitized and subsequently challenged with ovalbumin (OVA). Efficacy was determined by measuring both airway hyper-responsiveness (AHR) upon methacholine (MCh) challenge using whole body plethysmography and pulmonary eosinophilia by quantifying the numbers of these cells in the bronchoalveolar lavage fluid (BALF). Several other potential biomarkers of GSNOR inhibition were measured including levels of nitrite, cyclic guanosine monophosphate (cGMP), and inflammatory cytokines, as well as DNA binding activity of nuclear factor kappa B (NFκB). The dose response, onset of action, and duration of action of a single intravenous dose of N6022 given from 30 min to 48 h prior to MCh challenge were determined and compared to effects in mice not sensitized to OVA. The direct effect of N6022 on airway smooth muscle tone also was assessed in isolated rat tracheal rings. Results N6022 attenuated AHR (ED50 of 0.015 ± 0.002 mg/kg; Mean ± SEM) and eosinophilia. Effects were observed from 30 min to 48 h after treatment and were comparable to those achieved with three inhaled doses of ipratropium plus albuterol used as the positive control. N6022 increased BALF nitrite and plasma cGMP, while restoring BALF and plasma inflammatory markers toward baseline values. N6022 treatment also attenuated the OVA-induced increase in NFκB activation. In rat tracheal rings, N6022 decreased contractile responses to MCh. Conclusions The significant bronchodilatory and anti-inflammatory actions of N6022 in the airways are consistent with restoration of GSNO levels through GSNOR inhibition. GSNOR inhibition may offer a therapeutic approach for the treatment of asthma and other inflammatory lung diseases. N6022 is currently being evaluated in clinical trials for the treatment of inflammatory lung disease.

Due to their ubiquity in aquatic environments and their contribution to total biomass, especially in oligotrophic systems, cyanobacteria can be viewed as a proxy for primary productivity in both marine and fresh waters. In this chapter we describe the development and use of picocyanobacterial bioreporters to measure the bioavailability of nutrients that may constrain total photosynthesis in both lacustrine and marine systems. Issues pertaining to bioreporter construction, performance and field applications are discussed. Specifically, luminescent Synechococcus spp. and Synechocystis spp. bioreporters are described that allow the bioavailability of phosphorus, nitrogen and iron to be accurately measured in environmental samples.

Due to their ubiquity in aquatic environments and their contribution to total biomass, especially in oligotrophic systems, cyanobacteria can be viewed as a proxy for primary productivity in both marine and fresh waters. In this chapter we describe the development and use of picocyanobacterial bioreporters to measure the bioavailability of nutrients that may constrain total photosynthesis in both lacustrine and marine systems. Issues pertaining to bioreporter construction, performance and field applications are discussed. Specifically, luminescent Synechococcus spp. and Synechocystis spp. bioreporters are described that allow the bioavailability of phosphorus, nitrogen and iron to be accurately measured in environmental samples.