The impact of inotersen on Neuropathy Impairment Score in patients with hereditary transthyretin amyloidosis with polyneuropathy.

BACKGROUND: Patients with hereditary transthyretin amyloidosis (ATTRv) frequently experience symptoms of polyneuropathy (PN) that worsen over time and impair daily functioning. Previous analyses supported efficacy of inotersen, an antisense oligonucleotide, to slow neuropathic progression in patients with ATTRv-PN, as indicated by larger mean changes, relative to placebo, in total score and several subscales of the Neuropathy Impairment Score (NIS), and for the subset of NIS items specific to lower limbs (NIS-LL) for the overall study sample. A key objective of the current study was to evaluate efficacy of inotersen for slowing neuropathic progression in NIS/NIS-LL within key clinical subgroups of patients with ATTRv-PN. Additionally, for this study, responder definition (RD) thresholds were estimated for NIS/NIS-LL total and subscale scores, for the purpose of evaluating clinically meaningful benefit of inotersen at the individual patient-level. METHODS: Post hoc analyses used data from the NEURO-TTR phase 3 trial of inotersen in patients with ATTRv-PN (NCT01737398). Treatment differences in mean changes on NIS/NIS-LL total and subscale scores from baseline to week 65 were examined within patient subgroups defined by clinical characteristics. Anchor- and distribution-based approaches estimated RDs for NIS/NIS-LL scores, with responders defined as patients who did not experience clinically meaningful neuropathic progression. Responder analyses compared the proportion of patients classified as responders for each NIS/NIS-LL score between treatment arms. RESULTS: Within each patient subgroup, mean increases in NIS/NIS-LL total and muscle weakness subscales were significantly smaller after 65 weeks of treatment with inotersen compared to placebo. Similar patterns were observed for some, but not all, subgroups on NIS/NIS-LL reflex subscale scores. Recommended RDs were 8.1 points for NIS total and 4.7 points for NIS-LL total. Patients receiving inotersen for 65 weeks were significantly less likely than those receiving placebo to exhibit clinically meaningful increases on NIS/NIS-LL total, muscle weakness, and sensation subscales. CONCLUSIONS: This study supports previous evidence for efficacy of inotersen in this patient population and provides interpretation guidelines for clinically meaningful changes in NIS/NIS-LL scores.

The feasibility of using mouthpiece ventilation in the intensive care unit for post-extubation breathing support after acute tetraplegia.

STUDY DESIGN: A prospective cohort of patients with acute tetraplegia. OBJECTIVES: This study aimed to determine the feasibility of using mouthpiece ventilation (MPV) in the intensive care unit (ICU) for patients who are extubated after suffering an acute cervical spinal cord injury (CSCI). SETTING: ICU, Princess Alexandra Hospital, Brisbane Australia. METHODS: New admissions to ICU in the 14 months between April 2017 and June 2018 with a CSCI who underwent intubation were assessed for inclusion. MPV was provided to consenting participants (who were deemed likely to be able to maintain ventilation on their own) at the time of extubation and was utilised in addition to standard care while participants were awake. MPV settings, usage, and support hours to educate and facilitate MPV were collected. Feedback from participants and clinical staff was gathered throughout the study. Pre- and post-extubation measures of forced vital capacity (FVC), the frequency of endotracheal suction of sputum, and gas exchange using ventilation-perfusion ratios were recorded along with the incidence of reintubation. RESULTS: Fourteen participated in utilising MPV with 16 episodes of extubation. The average time per participant to have MPV titrated and bedside data collected was 178 minutes. Data from 16 episodes of extubation have been included. Three of the 14 participants failed initial extubation. Feedback from participants and clinicians has been positive and constructive, enabling MPV settings to be adapted to the person with acute CSCI during this pilot study. CONCLUSION: MPV is feasible to use post-extubation for people with CSCI in ICU. Pressure control mode MPV was deemed the most suitable for newly extubated acute CSCI patients. Intensive clinical support is required initially to provide education prior to MPV, and at the time of extubation for both patient and treating clinicians. Both report it to be a useful adjunct to ICU treatment.

Improving the handover of complex trauma patients by implementing a standardised process.

BACKGROUND: Patient handover continues to be an international health priority in the prevention of patient harm. Transitioning patients from the intensive care unit (ICU) to the ward is complex, particularly for trauma patients, due to the multifaceted aspects of their care requirements as a result of multiple injuries and different speciality teams. OBJECTIVES/AIM: To design, implement, and evaluate the efficacy of a standardised handover process and tool for the transfer of ICU trauma patients. METHODS: A multimethod before/after study design was used. This included observations before and after an implemented transfer process and semistructured interviews with ICU and ward nurses caring for trauma patients. Comparisons were made of data before and after the intervention. RESULTS: Eleven patient handovers were observed, and 21 nurses (11 from the ICU and 10 from the ward) were interviewed. Patients and family members were included during the handover following the intervention (n = 0/10 [0%] vs n = 4/11 [36%]) and the ward nurses were asked if they had any concerns (n = 5/10 [50%] vs n = 10/11 [91%]). Improvements in patient observations handed over were reported following the intervention. However, omissions remained in some key areas including patient introduction, patient identity, fluid balance, and allergies/alerts. Thematic analysis of interviews revealed that the new handover process was perceived advantageous by both ICU and ward nurses because of its structured and comprehensive approach. Identified future improvements included the need for hospital service managers to ensure integration of ICU and ward electronic health record systems. CONCLUSION: Precise, accurate, and complete handover remains a patient safety concern. Improvements were achieved using a standardised process and handover tool for the transfer of complex trauma patients. Further improvements are required to reduce the failure to hand over essential patient information.

Factors associated with increased health-related quality-of-life benefits in hereditary transthyretin amyloidosis polyneuropathy patients treated with inotersen.

INTRODUCTION/AIMS: Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a genetic condition associated with significant morbidity and mortality. In this study we aimed to identify patient subgroups exhibiting the greatest health-related quality of life (HRQL) benefit from inotersen treatment. METHODS: We examined data from the inotersen phase 2/3 randomized, controlled trial for ATTRv-PN, NEURO-TTR (NCT01737398, 66 weeks). LASSO regression models predicted changes in Norfolk QoL-DN total score (TQoL, range -4 to 136; higher scores indicate poorer HRQL) from baseline in the inotersen and placebo arm, respectively. Individualized efficacy scores (ES) were calculated as differences between predicted change scores had patients received inotersen vs placebo. Patients were ranked by ES to define the greatest-benefit subpopulation (top 50%). Characteristics of the top 50% and bottom 50% of patients were compared. RESULTS: The overall mean ± standard deviation TQoL change was -0.20 ± 19.13 for inotersen (indicating no change) and 10.77 ± 21.13 for placebo (indicating deterioration). Within the highest-benefit patients, mean TQoL change was -11.03 ± 17.06 (improvement) for inotersen and 11.24 ± 22.97 (deterioration) for placebo (P < .001). Compared with the overall population, patients in the greatest-benefit subpopulation were younger, more likely to have polyneuropathy disability (PND) scores 1 or 2, less likely to have received prior tafamidis or diflunisal treatment, and more likely to have Val30Met mutations and higher (worse) baseline TQoL. CONCLUSIONS: Patients who were younger and/or at earlier polyneuropathy stages experienced greater HRQL benefits from inotersen over 66 weeks. These findings underscore the need for early diagnosis and treatment initiation, especially among more severely affected patients in early stages of ATTRv-PN.

Long-term treatment effects of inotersen on health-related quality of life in patients with hATTR amyloidosis with polyneuropathy: Analysis of the open-label extension of the NEURO-TTR trial.

INTRODUCTION/AIMS: Hereditary transthyretin-mediated amyloidosis with polyneuropathy (hATTR-PN) progressively affects patients' functionality and compromises health-related quality of life (HRQL). The aim of this study was to quantify the projected long-term treatment effects of inotersen vs placebo on HRQL measures. METHODS: The inotersen phase 2/3 randomized, double-blind, placebo-controlled trial NEURO-TTR (NCT01737398, 65 weeks) and its subsequent open-label extension (OLE; NCT02175004, 104 weeks) included 172 (112 inotersen and 60 placebo) patients. Placebo double-blind period and overall inotersen-inotersen (double-blind/OLE) treatment period (170 weeks) data were used to extrapolate the long-term placebo-placebo effect using mixed-effects models with repeated measures. Changes from baseline in the Norfolk Quality of Life-Diabetic Neuropathy (QoL-DN) and 36-Item Short Form Health Survey version 2 (SF-36v2) in hATTR-PN were estimated. Differences in changes were compared between the inotersen-inotersen and extrapolated placebo-placebo arms. RESULTS: Inotersen-inotersen patients maintained their HRQL with an observed change ranging from 10.3% improvement (Norfolk QoL-DN item "Pain kept you awake at night") to 11.6% deterioration (SF-36v2 Activities of Daily Living subdomain). The extrapolated placebo-placebo results suggest greater deterioration over time compared with inotersen-inotersen treatment on Norfolk QoL-DN total score (23.6; 95% confidence interval [CI], 8.9-38.3; P < .01), Activities of Daily Living (4.6; 95% CI, 2.0-7.3; P < .001), and "Pain kept you awake at night" (1.2; 95% CI, 0.4-1.9; P < .01). Similarly, greater deterioration was expected for the SF-36v2 Physical Component Summary (8.0; 95% CI, 3.2-12.8, P < .01), Bodily Pain (7.8; 95% CI, 2.0-13.5; P < .01), and Physical Functioning (10.6; 95% CI, 5.5-15.6; P < .0001). DISCUSSION: Long-term (>3 years) inotersen treatment was associated with slowing and, in some domains, halting of deterioration in key HRQL outcome measures, particularly physical functioning and pain.

Rate of neuropathic progression in hereditary transthyretin amyloidosis with polyneuropathy and other peripheral neuropathies: a systematic review and meta-analysis.

BACKGROUND: We aimed to compare neuropathic progression rate between hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) and other peripheral neuropathies, including diabetic peripheral neuropathy (DPN) and Charcot-Marie-Tooth disease (CMT). METHODS: Literature searches identified studies reporting neuropathic progression, measured by Neuropathy Impairment Score (NIS) or NIS-Lower Limbs (NIS-LL). Our study also included unpublished data from a clinical registry of patients who were diagnosed with different peripheral neuropathies and seen at the Oregon Health & Science University (OHSU) during 2016-2020. Meta-analysis and meta-regression models examined and compared annual progression rates, calculated from extracted data, between studies of ATTRv-PN and other peripheral neuropathies. RESULTS: Data were synthesized from 15 studies in which NIS and/or NIS-LL total scores were assessed at least twice, with ≥12 weeks between assessments, among untreated patients with ATTRv-PN or other peripheral neuropathies. Meta-analysis models yielded that the annual progression rate in NIS total scores was significantly different from zero for studies in ATTRv-PN and CMT (11.77 and 1.41; both P < 0.001), but not DPN (- 1.96; P = 0.147). Meta-regression models showed significantly faster annual progression in studies in ATTRv-PN, which statistically exceeded that in other peripheral neuropathies by 12.45 points/year for NIS, and 6.96 for NIS-LL (both P < 0.001). CONCLUSIONS: Peripheral nervous function deteriorates more rapidly in patients with ATTRv-PN than for other peripheral neuropathies. These findings may improve understanding of the natural history of neuropathy in ATTRv-PN, facilitate early diagnosis, and guide the development of assessment tools and therapies specifically targeting neuropathic progression in this debilitating disease.

The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.

Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearly one-third were not previously annotated as RNA binding, and about 15% were not predictable by computational methods to interact with RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of mRNA binders with diverse molecular functions participating in combinatorial posttranscriptional gene-expression networks.

Handover practices of nurses transferring trauma patients from intensive care units to the ward: A multimethod observational study.

Poor-quality patient handover leads to adverse patient outcomes. Consequently, handover has been identified as a national and international priority for preventing patient harm. Risks are exacerbated during transfers of trauma intensive care unit (ICU) patients to a ward because of the complexity of their injuries coupled with a de-escalation in care and monitoring. This study assessed current handover practices for ICU trauma patients, identifying barriers and facilitators to best practice handover. A multimethod design was used, including naturalistic observations of clinical handover of trauma patients transferred to a ward and semistructured interviews with both the ICU and ward nurses caring for the trauma patient. The study was conducted at an Australian metropolitan public adult teaching hospital ICU. Purposive maximal sampling of patient handover opportunities was sought. Recruitment continued until data saturation was reached using thematic analysis. Ten ICU and ward nurses were recruited, with 10 observations of handover and 20 interviews conducted. Observations of the handovers identified multiple issues, including deficits and discrepancies in the information communicated that could impact patient safety, variable handover processes, and poor patient and family involvement. Interviews elicited two major themes around the handover: practices and processes. Nurses identified that interruptions, time, and workload pressures presented barriers to handover, whilst teamwork, using a structured and systematic approach, preparation time for handover, and communication before transfer facilitated effective handover and transfer. Nurses suggested a structured tool to aid handover. This study identified clinically significant deficits and discrepancies in the information communicated to the ward nurses. Nurses identified that interruptions, time, and workload pressures presented barriers to effective handover. Teamwork where preparation and the handover event are prioritised over other activities is needed. A minimum data set for handover in conjunction with patients and family members is recommended.

Tidal Deformabilities and Radii of Neutron Stars from the Observation of GW170817.

We use gravitational-wave observations of the binary neutron star merger GW170817 to explore the tidal deformabilities and radii of neutron stars. We perform a Bayesian parameter estimation with the source location and distance informed by electromagnetic observations. We also assume that the two stars have the same equation of state; we demonstrate that, for stars with masses comparable to the component masses of GW170817, this is effectively implemented by assuming that the stars' dimensionless tidal deformabilities are determined by the binary's mass ratio q by Λ_{1}/Λ_{2}=q^{6}. We investigate different choices of prior on the component masses of the neutron stars. We find that the tidal deformability and 90% credible interval is Λ[over ˜]=222_{-138}^{+420} for a uniform component mass prior, Λ[over ˜]=245_{-151}^{+453} for a component mass prior informed by radio observations of Galactic double neutron stars, and Λ[over ˜]=233_{-144}^{+448} for a component mass prior informed by radio pulsars. We find a robust measurement of the common areal radius of the neutron stars across all mass priors of 8.9≤R[over ^]≤13.2 km, with a mean value of ⟨R[over ^]⟩=10.8 km. Our results are the first measurement of tidal deformability with a physical constraint on the star's equation of state and place the first lower bounds on the deformability and areal radii of neutron stars using gravitational waves.

Erratum: Tidal Deformabilities and Radii of Neutron Stars from the Observation of GW170817 [Phys. Rev. Lett. 121, 091102 (2018)].

This corrects the article DOI: 10.1103/PhysRevLett.121.091102.

Honeybees use the skyline in orientation.

In view-based navigation, animals acquire views of the landscape from various locations and then compare the learned views with current views in order to orient in certain directions or move toward certain destinations. One landscape feature of great potential usefulness in view-based navigation is the skyline, the silhouette of terrestrial objects against the sky, as it is distant, relatively stable and easy to detect. The skyline has been shown to be important in the view-based navigation of ants, but no flying insect has yet been shown definitively to use the skyline in this way. Here, we show that honeybees do indeed orient using the skyline. A feeder was surrounded with an artificial replica of the natural skyline there, and the bees' departures toward the nest were recorded from above with a video camera under overcast skies (to eliminate celestial cues). When the artificial skyline was rotated, the bees' departures were rotated correspondingly, showing that the bees oriented by the artificial skyline alone. We discuss these findings in the context of the likely importance of the skyline in long-range homing in bees, the likely importance of altitude in using the skyline, the likely role of ultraviolet light in detecting the skyline, and what we know about the bees' ability to resolve skyline features.

Negative example selection for protein function prediction: the NoGO database.

Negative examples - genes that are known not to carry out a given protein function - are rarely recorded in genome and proteome annotation databases, such as the Gene Ontology database. Negative examples are required, however, for several of the most powerful machine learning methods for integrative protein function prediction. Most protein function prediction efforts have relied on a variety of heuristics for the choice of negative examples. Determining the accuracy of methods for negative example prediction is itself a non-trivial task, given that the Open World Assumption as applied to gene annotations rules out many traditional validation metrics. We present a rigorous comparison of these heuristics, utilizing a temporal holdout, and a novel evaluation strategy for negative examples. We add to this comparison several algorithms adapted from Positive-Unlabeled learning scenarios in text-classification, which are the current state of the art methods for generating negative examples in low-density annotation contexts. Lastly, we present two novel algorithms of our own construction, one based on empirical conditional probability, and the other using topic modeling applied to genes and annotations. We demonstrate that our algorithms achieve significantly fewer incorrect negative example predictions than the current state of the art, using multiple benchmarks covering multiple organisms. Our methods may be applied to generate negative examples for any type of method that deals with protein function, and to this end we provide a database of negative examples in several well-studied organisms, for general use (The NoGO database, available at: bonneaulab.bio.nyu.edu/nogo.html).

Parametric Bayesian priors and better choice of negative examples improve protein function prediction.

MOTIVATION: Computational biologists have demonstrated the utility of using machine learning methods to predict protein function from an integration of multiple genome-wide data types. Yet, even the best performing function prediction algorithms rely on heuristics for important components of the algorithm, such as choosing negative examples (proteins without a given function) or determining key parameters. The improper choice of negative examples, in particular, can hamper the accuracy of protein function prediction. RESULTS: We present a novel approach for choosing negative examples, using a parameterizable Bayesian prior computed from all observed annotation data, which also generates priors used during function prediction. We incorporate this new method into the GeneMANIA function prediction algorithm and demonstrate improved accuracy of our algorithm over current top-performing function prediction methods on the yeast and mouse proteomes across all metrics tested. AVAILABILITY: Code and Data are available at: http://bonneaulab.bio.nyu.edu/funcprop.html

siRNA-mediated knockdown of P450 oxidoreductase in rats: a tool to reduce metabolism by CYPs and increase exposure of high clearance compounds.

PURPOSE: To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. METHODS: siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. RESULTS: Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. CONCLUSIONS: Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.

Mood impairments in adults previously diagnosed with developmental coordination disorder.

BACKGROUND: Developmental coordination disorder (DCD) affects up to 6% of the population and is diagnosed on the basis of poor motor coordination. While we know rather little about its lifetime consequences, clear and significant difficulties remain through the lifespan for the majority. Reduced physical activity and, outside of the motor domain, significant mental health issues exist for many with DCD. AIMS: This study provides the first investigation of the presence of mood disorders in adults with DCD. METHOD: Symptoms of anxiety and depression were assessed using the Beck Depression and Spielberger Anxiety Inventories in 36 adults previously diagnosed with DCD vs. 49 age- and gender-matched typical controls. Amount and type of physical activity undertaken each week were also reported. RESULTS: After controlling for their reduced level of weekly physical activity, the group with DCD reported significantly more symptoms of depression, state and trait anxiety than their peers. CONCLUSIONS: This finding has important implications for consideration of intervention in DCD, as well as for investigation of the risk and protective factors at play in long-term outcome. Finally, the findings highlight the need for awareness of motor difficulties in those presenting with high levels of anxiety and depression, and vice versa.

Responder analysis for neuropathic impairment and quality-of-life assessment in patients with hereditary transthyretin amyloidosis with polyneuropathy in the NEURO-TTR study.

OBJECTIVE: Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a rare disease characterized by rapid neuropathic progression. In pivotal studies of gene-silencing treatments, the modified Neuropathy Impairment Score + 7 tests (mNIS + 7) and Norfolk-Quality of Life (QOL)-Diabetic Neuropathy (DN) questionnaire assessed treatment impact on neuropathic progression. Establishing responder definition (RD) thresholds for these measures would enable evaluation of clinically meaningful treatment benefit. METHODS: mNIS + 7 and Norfolk-QOL-DN were administered at baseline and week 65 to 165 adults with ATTRv-PN receiving inotersen (n = 106) or placebo (n = 59) in the NEURO-TTR study. Anchor-based approaches for estimating RD thresholds were used for Norfolk QOL-DN, while distribution-based approaches were used for both measures. Responders were patients with a score change < RD, indicating improvement or stabilization (i.e., no clinically meaningful progression). Odds ratios (ORs) and Fisher's exact tests compared proportions of responders by treatment. RESULTS: The mean RD estimates were 12.2 points and 8.8 points for mNIS + 7 and Norfolk QOL-DN, respectively. The proportions of patients whose change in score indicated improvement or stabilization were statistically significantly larger for inotersen than placebo for all estimated RD thresholds for mNIS + 7 (64-86% responders for inotersen vs. 27-46% for placebo, ORs = 3.8-7.2, ps < 0.001) and Norfolk QOL-DN (66-81% vs. 35-56%, ORs = 2.4-3.6, ps < 0.05). DISCUSSION: Establishing RD thresholds for these instruments enables evaluation of clinically relevant and individual-level treatment benefit on neuropathic progression. Across RDs estimated using multiple methods, a higher proportion of patients receiving inotersen than placebo showed improved or stabilized neuropathic progression at week 65. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01737398; Date of registration: November 29, 2012.

Clinical manifestations and healthcare utilization before diagnosis of transthyretin amyloidosis.

Introduction: Initial clinical manifestations of transthyretin amyloidosis (ATTR) are not well understood, making timely diagnosis challenging. Methods: Patients aged ≥68 years newly diagnosed with ATTR were identified using Medicare Research Identifiable Files. Symptom manifestation and healthcare utilization were measured during 3 years pre-diagnosis; demographics and comorbidity index during 1-year pre-diagnosis. Controls (ATTR-free) were matched 1:1 to patients with ATTR based on age, sex and region; same index date and enrollment as match. Results: We identified 552 matched ATTR-control pairs: mean age 78.3 (standard deviation 6.3) and 64.5% male. Among patients with ATTR (vs controls), cardiovascular conditions (92.9 vs 75.9%) and hospitalization (54.0 vs 35.5%) were frequent during 3 years pre-diagnosis. Conclusion: Patients with ATTR have multiple symptoms and hospitalizations pre-diagnosis, recognition of which may facilitate earlier diagnosis and treatment.

Developing antisense oligonucleotides for a TECPR2 mutation-induced, ultra-rare neurological disorder using patient-derived cellular models.

Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with ∼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.

Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats.

The interaction between the gut microbiota and their mammalian host is known to have far-reaching consequences with respect to metabolism and health. We investigated the effects of eight days of oral antibiotic exposure (penicillin and streptomycin sulfate) on gut microbial composition and host metabolic phenotype in male Han-Wistar rats (n = 6) compared to matched controls. Early recolonization was assessed in a third group exposed to antibiotics for four days followed by four days recovery (n = 6). Fluorescence in situ hybridization analysis of the intestinal contents collected at eight days showed a significant reduction in all bacterial groups measured (control, 10(10.7) cells/g feces; antibiotic-treated, 10(8.4)). Bacterial suppression reduced the excretion of mammalian-microbial urinary cometabolites including hippurate, phenylpropionic acid, phenylacetylglycine and indoxyl-sulfate whereas taurine, glycine, citrate, 2-oxoglutarate, and fumarate excretion was elevated. While total bacterial counts remained notably lower in the recolonized animals (10(9.1) cells/g faeces) compared to the controls, two cage-dependent subgroups emerged with Lactobacillus/Enterococcus probe counts dominant in one subgroup. This dichotomous profile manifested in the metabolic phenotypes with subgroup differences in tricarboxylic acid cycle metabolites and indoxyl-sulfate excretion. Fecal short chain fatty acids were diminished in all treated animals. Antibiotic treatment induced a profound effect on the microbiome structure, which was reflected in the metabotype. Moreover, the recolonization process was sensitive to the microenvironment, which may impact on understanding downstream consequences of antibiotic consumption in human populations.