Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU): Trial Satisfaction and Attitudes towards Future Clinical Trials.

BACKGROUND: Clinical trial satisfaction is increasingly important for future trial designs and is associated with treatment adherence and willingness to enroll in future research studies or to recommend trial participation. In this post-trial survey, we examined participant satisfaction and attitudes toward future clinical trials in the Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU). METHODS: We developed an anonymous, participant satisfaction survey tailored to participants enrolled in the DIAN-TU-001 double-blind clinical trial of solanezumab or gantenerumab and requested that all study sites share the survey with their trial participants. A total of 194 participants enrolled in the trial at 24 study sites. We utilized regression analysis to explore the link between participants' clinical trial experiences, their satisfaction, and their willingness to participate in upcoming trials. RESULTS: Survey responses were received over a sixteen-month window during 2020-2021 from 58 participants representing 15 study sites. Notably, 96.5% of the survey respondents expressed high levels of satisfaction with the trial, 91.4% would recommend trial participation, and 96.5% were willing to enroll again. Age, gender, and education did not influence satisfaction levels. Participants reported enhanced medical care (70.7%) and pride in contributing to the DIAN-TU trial (84.5%). Satisfaction with personnel and procedures was high (98.3%). Respondents had a mean age of 48.7 years, with most being from North America and Western Europe, matching the trial's demographic distribution. Participants' decisions to learn their genetic status increased during the trial, and most participants endorsed considering future trial participation regardless of the DIAN-TU-001 trial outcome. CONCLUSION: Results suggest that DIAN-TU-001 participants who responded to the survey exhibited high motivation to participate in research, overall satisfaction with the clinical trial, and willingness to participate in research in the future, despite a long trial duration of 4-7 years with detailed annual clinical, cognitive, PET, MRI, and lumbar puncture assessments. Implementation of features that alleviate barriers and challenges to trial participation is like to have a high impact on trial satisfaction and reduce participant burden.

Miniaturized engineered heart tissues from hiPSC-derived triple cell type co-cultures to study human cardiac function.

Human heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better than standard two-dimensional models in vitro. They typically consist of less than 5000 cells and are used to measure contraction kinetics although not contraction force. By contrast, engineered heart tissues (EHTs) formed around two flexible pillars, can measure contraction force but conventional EHTs often require between 0.5 and 2 million cells. This makes large-scale screening of many EHTs costly. Our goals here were (i) to create a physiologically relevant model that required fewer cells than standard EHTs making them less expensive, and (ii) to ensure that this miniaturized model retained correct functionality. We demonstrated that fully functional EHTs could be generated from physiologically relevant combinations of hiPSC-derived cardiomyocytes (70%), cardiac fibroblasts (15%) and cardiac endothelial cells (15%), using as few as 1.6 × 104 cells. Our results showed that these EHTs were viable and functional up to 14 days after formation. The EHTs could be electrically paced in the frequency range between 0.6 and 3 Hz, with the optimum between 0.6 and 2 Hz. This was consistent across three downscaled EHT sizes tested. These findings suggest that miniaturized EHTs could represent a cost-effective microphysiological system for disease modelling and examining drug responses particularly in secondary screens for drug discovery.

Investigating Partially Discordant Results in Phase 3 Studies of Aducanumab.

OBJECTIVES: Efficacy and safety results from the EMERGE (NCT02484547) and ENGAGE (NCT02477800) phase 3 studies of aducanumab in early Alzheimer's disease (AD) have been published. In EMERGE, but not in ENGAGE, high-dose aducanumab demonstrated significant treatment effects across primary and secondary endpoints. Low-dose aducanumab results were consistent across studies with non-significant differences versus placebo that were intermediate to the high-dose arm in EMERGE. The present investigation examined data from EMERGE and ENGAGE through post-hoc analyses to determine factors that contributed to discordant results between the high-dose arms of the two studies. DESIGN: EMERGE and ENGAGE were 2 phase 3, randomized, double-blind, placebo-controlled, parallel-group studies. SETTING: EMERGE and ENGAGE were 2 global multicenter studies involving 348 sites in 20 countries. PARTICIPANTS: Participants in EMERGE and ENGAGE were aged 50 to 85 years and had mild cognitive impairment or mild AD dementia with confirmed amyloid pathology. The randomized and dosed population (all randomized patients who received at least one dose of study treatment) included 1638 patients in EMERGE and 1647 in ENGAGE. INTERVENTION: In EMERGE and ENGAGE, participants were randomized to receive low- or high-dose aducanumab or placebo (1:1:1) once every 4 weeks. MEASUREMENTS: In this paper, 4 areas were investigated through post-hoc analyses to understand the discordance in the high-dose arms of the EMERGE and ENGAGE studies: baseline characteristics, amyloid-related imaging abnormalities, non-normality of the data, and dosing/exposure to aducanumab. RESULTS: Post-hoc analyses showed that outcomes in the ENGAGE high-dose group were affected by an imbalance in a small number of patients with extremely rapid progression and by lower exposure to the target dose of 10 mg/kg. These factors were confounded and present in early enrolled patients but were not present in later-enrolled patients who were randomized to the target dosing regimen of 10 mg/kg after titration. Neither baseline characteristics nor amyloid-related imaging abnormalities contributed to the difference in results between the high-dose arms. CONCLUSIONS: Results were consistent across studies in later enrolled patients in which the incidence of rapidly progressing patients was balanced across treatment arms.

Introduction of a Geminin mScarlet Reporter into H2B-mTurq2 hiPSCs for Live-cell Imaging of Proliferation and Cell Cycling.

We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ubiquitously transcribed mScarletI-Geminin (GMMN) (1-110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.

Two Randomized Phase 3 Studies of Aducanumab in Early Alzheimer's Disease.

BACKGROUND: Alzheimer's disease is a progressive, irreversible, and fatal disease for which accumulation of amyloid beta is thought to play a key role in pathogenesis. Aducanumab is a human monoclonal antibody directed against aggregated soluble and insoluble forms of amyloid beta. OBJECTIVES: We evaluated the efficacy and safety of aducanumab in early Alzheimer's disease. DESIGN: EMERGE and ENGAGE were two randomized, double-blind, placebo-controlled, global, phase 3 studies of aducanumab in patients with early Alzheimer's disease. SETTING: These studies involved 348 sites in 20 countries. PARTICIPANTS: Participants included 1638 (EMERGE) and 1647 (ENGAGE) patients (aged 50-85 years, confirmed amyloid pathology) who met clinical criteria for mild cognitive impairment due to Alzheimer's disease or mild Alzheimer's disease dementia, of which 1812 (55.2%) completed the study. INTERVENTION: Participants were randomly assigned 1:1:1 to receive aducanumab low dose (3 or 6 mg/kg target dose), high dose (10 mg/kg target dose), or placebo via IV infusion once every 4 weeks over 76 weeks. MEASUREMENTS: The primary outcome measure was change from baseline to week 78 on the Clinical Dementia Rating Sum of Boxes (CDR-SB), an integrated scale that assesses both function and cognition. Other measures included safety assessments; secondary and tertiary clinical outcomes that assessed cognition, function, and behavior; and biomarker endpoints. RESULTS: EMERGE and ENGAGE were halted based on futility analysis of data pooled from the first approximately 50% of enrolled patients; subsequent efficacy analyses included data from a larger data set collected up to futility declaration and followed prespecified statistical analyses. The primary endpoint was met in EMERGE (difference of -0.39 for high-dose aducanumab vs placebo [95% CI, -0.69 to -0.09; P=.012; 22% decrease]) but not in ENGAGE (difference of 0.03, [95% CI, -0.26 to 0.33; P=.833; 2% increase]). Results of biomarker substudies confirmed target engagement and dose-dependent reduction in markers of Alzheimer's disease pathophysiology. The most common adverse event was amyloid-related imaging abnormalities-edema. CONCLUSIONS: Data from EMERGE demonstrated a statistically significant change across all four primary and secondary clinical endpoints. ENGAGE did not meet its primary or secondary endpoints. A dose- and time-dependent reduction in pathophysiological markers of Alzheimer's disease was observed in both trials.

Generation of LUMCi041-A-2: Equipping a PAX3 reporter iPSC line with doxycycline inducible H2B-mTurquoise2 for live cell imaging.

An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end we introduced a 3rd generation "all-in-one" doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells. Besides being a universal reporter in iPSC-based differentiation and toxicity assays, the generated pluripotent and genomically normal LUMCi041-A-2 line is particularly suited to study PAX3-positive stages of development.

Predictors for a dementia gene mutation based on gene-panel next-generation sequencing of a large dementia referral series.

Next-generation genetic sequencing (NGS) technologies facilitate the screening of multiple genes linked to neurodegenerative dementia, but there are few reports about their use in clinical practice. Which patients would most profit from testing, and information on the likelihood of discovery of a causal variant in a clinical syndrome, are conspicuously absent from the literature, mostly for a lack of large-scale studies. We applied a validated NGS dementia panel to 3241 patients with dementia and healthy aged controls; 13,152 variants were classified by likelihood of pathogenicity. We identified 354 deleterious variants (DV, 12.6% of patients); 39 were novel DVs. Age at clinical onset, clinical syndrome and family history each strongly predict the likelihood of finding a DV, but healthcare setting and gender did not. DVs were frequently found in genes not usually associated with the clinical syndrome. Patients recruited from primary referral centres were compared with those seen at higher-level research centres and a national clinical neurogenetic laboratory; rates of discovery were comparable, making selection bias unlikely and the results generalisable to clinical practice. We estimated penetrance of DVs using large-scale online genomic population databases and found 71 with evidence of reduced penetrance. Two DVs in the same patient were found more frequently than expected. These data should provide a basis for more informed counselling and clinical decision making.

Interpretation of field potentials measured on a multi electrode array in pharmacological toxicity screening on primary and human pluripotent stem cell-derived cardiomyocytes.

Multi electrode arrays (MEAs) are increasingly used to detect external field potentials in electrically active cells. Recently, in combination with cardiomyocytes derived from human (induced) pluripotent stem cells they have started to become a preferred tool to examine newly developed drugs for potential cardiac toxicity in pre-clinical safety pharmacology. The most important risk parameter is proarrhythmic activity in cardiomyocytes which can cause sudden cardiac death. Whilst MEAs can provide medium- to high- throughput noninvasive assay platform, the translation of a field potential to cardiac action potential (normally measured by low-throughput patch clamp) is complex so that accurate assessment of drug risk to the heart is in practice still challenging. To address this, we used computational simulation to study the theoretical relationship between aspects of the field potential and the underlying cardiac action potential. We then validated the model in both primary mouse- and human pluripotent (embryonic) stem cell-derived cardiomyocytes showing that field potentials measured in MEAs could be converted to action potentials that were essentially identical to those determined directly by electrophysiological patch clamp. The method significantly increased the amount of information that could be extracted from MEA measurements and thus combined the advantages of medium/high throughput with more informative readouts. We believe that this will benefit the analysis of drug toxicity screening of cardiomyocytes using in time and accuracy.

Human heart disease: lessons from human pluripotent stem cell-derived cardiomyocytes.

Technical advances in generating and phenotyping cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are now driving their wider acceptance as in vitro models to understand human heart disease and discover therapeutic targets that may lead to new compounds for clinical use. Current literature clearly shows that hPSC-CMs recapitulate many molecular, cellular, and functional aspects of human heart pathophysiology and their responses to cardioactive drugs. Here, we provide a comprehensive overview of hPSC-CMs models that have been described to date and highlight their most recent and remarkable contributions to research on cardiovascular diseases and disorders with cardiac traits. We conclude discussing immediate challenges, limitations, and emerging solutions.

Small molecule absorption by PDMS in the context of drug response bioassays.

The polymer polydimethylsiloxane (PDMS) is widely used to build microfluidic devices compatible with cell culture. Whilst convenient in manufacture, PDMS has the disadvantage that it can absorb small molecules such as drugs. In microfluidic devices like "Organs-on-Chip", designed to examine cell behavior and test the effects of drugs, this might impact drug bioavailability. Here we developed an assay to compare the absorption of a test set of four cardiac drugs by PDMS based on measuring the residual non-absorbed compound by High Pressure Liquid Chromatography (HPLC). We showed that absorption was variable and time dependent and not determined exclusively by hydrophobicity as claimed previously. We demonstrated that two commercially available lipophilic coatings and the presence of cells affected absorption. The use of lipophilic coatings may be useful in preventing small molecule absorption by PDMS.

A panel of nine cerebrospinal fluid biomarkers may identify patients with atypical parkinsonian syndromes.

BACKGROUND: Patients presenting with parkinsonian syndromes share many clinical features, which can make diagnosis difficult. This is important as atypical parkinsonian syndromes (APSs) such as progressive supranuclear palsy (PSP), multiple system atrophy (MSA) and corticobasal syndrome (CBS) carry a poor prognosis, compared with patients with Parkinson's disease (PD). In addition, there is overlap between APS and dementia diseases, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD). OBJECTIVE: To use a panel of cerebrospinal fluid (CSF) biomarkers to differentiate patients with APS from PD and dementia. METHODS: A prospective cohort of 160 patients and 30 control participants were recruited from a single specialist centre. Patients were clinically diagnosed according to current consensus criteria. CSF samples were obtained from patients with clinical diagnoses of PD (n=31), PSP (n=33), CBS (n=14), MSA (n=31), AD (n=26) and FTD (n=16). Healthy, elderly participants (n=30) were included as controls. Total τ (t-τ), phosphorylated τ (p-τ), ß-amyloid 1-42 (Aß42), neurofilament light chain (NFL), α-synuclein (α-syn), amyloid precursor protein soluble metabolites α and ß (soluble amyloid precursor protein (sAPP)α, sAPPß) and two neuroinflammatory markers (monocyte chemoattractant protein-1 and YKL-40) were measured in CSF. A reverse stepwise regression analysis and the false discovery rate procedure were used. RESULTS: CSF NFL (p<0.001), sAPPα (p<0.001) and a-syn (p=0.003) independently predicted diagnosis of PD versus APS. Together, these nine biomarkers could differentiate patients with PD from APS with an area under the curve of 0.95 and subtypes of APS from one another. There was good discriminatory power between parkinsonian groups, dementia disorders and healthy controls. CONCLUSIONS: A panel of nine CSF biomarkers was able to differentiate APS from patients with PD and dementia. This may have important clinical utility in improving diagnostic accuracy, allowing better prognostication and earlier access to potential disease-modifying therapies.

Follow-up of Thalidomide treatment in patients with Hereditary Haemorrhagic Telangiectasia.

BACKGROUND: Patients with a hereditary vascular disorder called Rendu-Osler-Weber syndrome (Hereditary Haemorrhagic Telangiectasia, HHT) haemorrhage easily due to weak-walled vessels. Haemorrhage in lungs or brain can be fatal but patients suffer most from chronic and prolonged nosebleeds (epistaxis), the frequency and intensity of which increases with age. Several years ago, it was discovered serendipitously that the drug Thalidomide had beneficial effects on the disease symptoms in several of a small group of HHT patients: epistaxis and the incidence of anaemia were reduced and patients required fewer blood transfusions. In addition, they reported a better quality of life. However, Thalidomide has significant negative side effects, including neuropathy and fatigue. METHODS: We followed up all HHT patients in the Netherlands who had been taking Thalidomide at the time the original study was completed to find out (i) how many had continued taking Thalidomide and for how long (ii) the nature and severity of any side-effects and (iii) whether side-effects had influenced their decision to continue taking Thalidomide. RESULTS: Only a minority of patients had continued taking the drug despite its beneficial effects on their symptoms and that the side effects were the primary reason to stop. CONCLUSION: Despite symptom reduction, alternative treatments are still necessary for epistaxis in HHT patients and a large-scale clinical trial is not justified although incidental use in the most severely affected patients can be considered.

Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage, blood outgrowth endothelial cells and cells from milk teeth.

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g., for children and minors) dental pulp cells from milk teeth represent a valuable alternative.

Repolarization reserve determines drug responses in human pluripotent stem cell derived cardiomyocytes.

Unexpected induction of arrhythmias in the heart is still one of the major risks of new drugs despite recent improvements in cardiac safety assays. Here we address this in a novel emerging assay system. Eleven reference compounds were administrated to spontaneously beating clusters of cardiomyocytes from human pluripotent stem cells (hPSC-CM) and the responses determined using multi-electrode arrays. Nine showed clear dose-dependence effects on field potential (FP) duration. Of these, the Ca(2+) channel blockers caused profound shortening of action potentials, whereas the classical hERG blockers, like dofetilide and d,l-sotalol, induced prolongation, as expected. Unexpectedly, two potent blockers of the slow component of the delayed rectifier potassium current (I(Ks)), HMR1556 and JNJ303, had only minor effects on the extracellular FP of wild-type hPSC-CM despite evidence of functional I(Ks) channels. These compounds were therefore re-evaluated under conditions that mimicked reduced "repolarization reserve," a parameter reflecting the capacity of cardiomyocytes to repolarize and a strong risk factor for the development of ventricular arrhythmias. Strikingly, in both pharmacological and genetic models of diminished repolarization reserve, HMR1556 and JNJ03 strongly increased the FP duration. These profound effects indicate that I(Ks) plays an important role in limiting action potential prolongation when repolarization reserve is attenuated. The findings have important clinical implications and indicate that enhanced sensitization to repolarization-prolonging compounds through pharmacotherapy or genetic predisposition should be taken into account when assessing drug safety.

Stem cell self-renewal: lessons from bone marrow, gut and iPS toward clinical applications.

The hematopoietic stem cell (HSC) is the prototype organ-regenerating stem cell (SC), and by far the most studied type of SC in the body. Currently, HSC-based therapy is the only routinely used SC therapy; however, advances in the field of embryonic SCs and induced pluripotent SCs may change this situation. Interest into in vitro generation of HSCs, including signals for HSC expansion and differentiation from these more primitive SCs, as well as advances in other organ-specific SCs, in particular the intestine, provide promising new applications for SC therapies. Here, we review the basic principles of different SC systems, and on the basis of the experience with HSC-based SC therapy, provide recommendations for clinical application of emerging SC technologies.

The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands.

One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.).