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Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or live-cell reporter tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the required components into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of CRISPR-Cas9 nucleases results in selection-free and precise whole transgene insertion in large fractions of target-cell populations (i.e. up to 93%) and disclose that programmable nuclease-induced chromosomal breaks promote AAV transduction. Finally, besides investigating relationships between distinct AAV structures and genome-editing performance endpoints, we further report that high-fidelity CRISPR-Cas9 nucleases are critical for mitigating off-target chromosomal insertion of defective AAV genomes known to be packaged in vector particles., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro , ex vivo , and in vivo , we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications., Competing Interests: Conflict of interest The authors declare that they have no competing interests.

Prime editors have high potential for disease modelling and regenerative medicine efforts including those directed at the muscle-wasting disorder Duchenne muscular dystrophy (DMD). However, the large size and multicomponent nature of prime editing systems pose substantial production and delivery issues. Here, we report that packaging optimized full-length prime editing constructs in adenovector particles (AdVPs) permits installing precise DMD edits in human myogenic cells, namely, myoblasts and mesenchymal stem cells (up to 80% and 64%, respectively). AdVP transductions identified optimized prime-editing reagents capable of correcting DMD reading frames of ∼14% of patient genotypes and restoring dystrophin synthesis and dystrophin-β-dystroglycan linkages in unselected DMD muscle cell populations. AdVPs were equally suitable for correcting DMD iPSC-derived cardiomyocytes and delivering dual prime editors tailored for DMD repair through targeted exon 51 deletion. Moreover, by exploiting the cell cycle-independent AdVP transduction process, we report that 2- and 3-component prime-editing modalities are both most active in cycling than in post-mitotic cells. Finally, we establish that combining AdVP transduction with seamless prime editing allows for stacking chromosomal edits through successive delivery rounds. In conclusion, AdVPs permit versatile investigation of advanced prime editing systems independently of their size and component numbers, which should facilitate their screening and application., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Advanced myelodysplastic syndrome (MDS) and juvenile myelomonocytic leukemia (JMML) are rare hematological malignancies in children. A second allograft is recommended if a relapse occurs after hematopoietic stem cell transplantation, but the outcome is poor., Objective: We conducted a phase I/II multicenter study to evaluate the safety, pharmacokinetics, and activity of azacitidine in children with relapsed MDS/JMML prior to the second hematopoietic stem cell transplantation., Methods: Patients enrolled from June 2013 to March 2019 received azacitidine intravenously/subcutaneously once daily on days 1-7 of a 28-day cycle. The MDS and JMML cohorts followed a two-stage design separately, with a safety run-in for JMML. Response and safety data were used to evaluate efficacy and establish the recommended dose. Pharmacokinetics was also analyzed. The study closed prematurely because of low recruitment., Results: Six patients with MDS and four patients with JMML received a median of three and five cycles, respectively. Azacitidine 75 mg/m 2 was well tolerated and plasma concentration-time profiles were similar to observed in adults. The most prevalent grade 3-4 adverse event was myelotoxicity. No responses were seen in patients with MDS, but 83% achieved stable disease; four patients underwent an allotransplant. Overall response rate in the JMML cohort was 75% (two complete responses; one partial response) and all responders underwent hematopoietic stem cell transplantation. One-year overall survival was 67% (95% confidence interval 38-100) in MDS and 50% (95% confidence interval 19-100) in JMML., Conclusions: Azacitidine 75 mg/m 2 prior to a second hematopoietic stem cell transplantation is safe in children with relapsed MDS/JMML. Although the long-term advantage remains to be assessed, this study suggests that azacitidine is an efficacious option for relapsed JMML., Clinical Trial Registration: EudraCT 2010-022235-10., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background and Objective: As a result of changes in physiology during pregnancy, the pharmacokinetics (PK) of drugs can be altered. It is unclear whether under- or overexposure occurs in pregnant cancer patients and thus also whether adjustments in dosing regimens are required. Given the severity of the malignant disease and the potentially high impact on both the mother and child, there is a high unmet medical need for adequate and tolerable treatment of this patient population. We aimed to develop and evaluate a semi-physiological enriched model that incorporates physiological changes during pregnancy into available population PK models developed from non-pregnant patient data., Methods: Gestational changes in plasma protein levels, renal function, hepatic function, plasma volume, extracellular water and total body water were implemented in existing empirical PK models for docetaxel, paclitaxel, epirubicin and doxorubicin. These models were used to predict PK profiles for pregnant patients, which were compared with observed data obtained from pregnant patients., Results: The observed PK profiles were well described by the model. For docetaxel, paclitaxel and doxorubicin, an overprediction of the lower concentrations was observed, most likely as a result of a lack of data on the gestational changes in metabolizing enzymes. For paclitaxel, epirubicin and doxorubicin, the semi-physiological enriched model performed better in predicting PK in pregnant patients compared with a model that was not adjusted for pregnancy-induced changes., Conclusion: By incorporating gestational changes into existing population pharmacokinetic models, it is possible to adequately predict plasma concentrations of drugs in pregnant patients which may inform dose adjustments in this population., (© 2023. The Author(s).)

Introduction: Dabrafenib and trametinib are currently administered at fixed doses, at which interpatient variability in exposure is high. The aim of this study was to investigate whether drug exposure is related to efficacy and toxicity in a real-life cohort of melanoma patients treated with dabrafenib plus trametinib., Patients and Methods: An observational study was performed in which pharmacokinetic samples were collected as routine care. Using estimated dabrafenib Area Under the concentration-time Curve and trametinib trough concentrations (C min ), univariable and multivariable exposure-response analyses were performed., Results: In total, 140 patients were included. Dabrafenib exposure was not related to either progression-free survival (PFS) or overall survival (OS). Trametinib exposure was related to survival, with C min  ≥ 15.6 ng/mL being identified as the optimal threshold. Median OS was significantly longer in patients with trametinib C min  ≥ 15.6 ng/mL (22.8 vs. 12.6 months, P = 0.003), with a multivariable hazard ratio of 0.55 (95% CI 0.36-0.85, P = 0.007). Median PFS in patients with trametinib C min levels ≥ 15.6 ng/mL (37%) was 10.9 months, compared with 6.0 months for those with C min below this threshold (P = 0.06). Multivariable analysis resulted in a hazard ratio of 0.70 (95% CI 0.47-1.05, P = 0.082). Exposure to dabrafenib and trametinib was not related to clinically relevant toxicities., Conclusions: Overall survival of metastasized melanoma patients with trametinib C min levels ≥ 15.6 ng/mL is ten months longer compared to patients with C min below this threshold. This would theoretically provide a rationale for therapeutic drug monitoring of trametinib. Although a high proportion of patients are underexposed, there is very little scope for dose increments due to the risk of serious toxicity., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs), in contrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genome editing based on dual CRISPR-Cas9 complexes (multiplexes) permits removing specific genomic sequences in living cells leveraging research on functional genomics and genetic therapies. Delivering the required large and multicomponent reagents in a synchronous and stoichiometric manner remains, however, challenging. Moreover, uncoordinated activity of independently acting CRISPR-Cas9 multiplexes increases the complexity of genome editing outcomes. Here, we investigate the potential of fostering precise multiplexing genome editing using high-capacity adenovector particles (AdVPs) for the delivery of Cas9 ortholog fusion constructs alone (forced Cas9 heterodimers) or together with their cognate guide RNAs (forced CRISPR-Cas9 heterodimers). We demonstrate that the efficiency and accuracy of targeted chromosomal DNA deletions achieved by single AdVPs encoding forced CRISPR-Cas9 heterodimers is superior to that obtained when the various components are delivered separately. Finally, all-in-one AdVP delivery of forced CRISPR-Cas9 heterodimers triggers robust DMD exon 51 splice site excision resulting in reading frame restoration and selection-free detection of dystrophin in muscle cells derived from Duchenne muscular dystrophy patients. In conclusion, AdVPs promote precise multiplexing genome editing through the integrated delivery of forced CRISPR-Cas9 heterodimer components, which, in comparison with split conventional CRISPR-Cas9 multiplexes, engage target sequences in a more coordinated fashion., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Correlations between increasing concentrations of circulating tumor DNA (ctDNA) in plasma and disease progression have been shown. A nonlinear mixed effects model to describe the dynamics of epidermal growth factor receptor (EGFR) ctDNA data from patients with non-small cell lung cancer (NSCLC) combined with a parametric survival model were developed to evaluate the ability of these modeling techniques to describe ctDNA data. Repeated ctDNA measurements on L858R, exon19del, and T790M mutants were available from 54 patients with EGFR mutated NSCLC treated with erlotinib or gefitinib. Different dynamic models were tested to describe the longitudinal ctDNA concentrations of the driver and resistance mutations. Subsequently, a parametric time-to-event model for progression-free survival (PFS) was developed. Predicted L858R, exon19del, and T790M concentrations were used to evaluate their value as predictor for disease progression. The ctDNA dynamics were best described by a model consisting of a zero-order increase and first-order elimination (19.7/day, 95% confidence interval [CI] 14.9-23.6/day) of ctDNA concentrations. In addition, time-dependent development of resistance (5.0 × 10 -4 , 95% CI 2.0 × 10 -4 -7.0 × 10 -4 /day) was included in the final model. Relative change in L858R and exon19del concentrations from baseline was identified as most significant predictor of disease progression (p = 0.001). The dynamic model for L858R, exon19del, and T790M concentrations in ctDNA and time-to-event model adequately described the observed concentrations and PFS data in our clinical cohort. In addition, it was shown that nonlinear mixed effects modeling is a valuable method for the analysis of longitudinal and heterogeneous biomarker datasets obtained from clinical practice., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Neurofibromatosis Type 1 (NF1) is a common genetic disorder frequently associated with cognitive deficits. Despite cognitive deficits being a key feature of NF1, the profile of such impairments in NF1 has been shown to be heterogeneous. Thus, we sought to quantitatively synthesize the extant literature on cognitive functioning in NF1. A random-effects meta-analysis of cross-sectional studies was carried out comparing cognitive functioning of patients with NF1 to typically developing or unaffected sibling comparison subjects of all ages. Analyses included 50 articles (Total N NF1 = 1,522; M Age = 15.70 years, range = 0.52-69.60), yielding 460 effect sizes. Overall moderate deficits were observed [g = -0.64, 95% CI = (-0.69, -0.60)] wherein impairments differed at the level of cognitive domain. Deficits ranged from large [general intelligence: g = -0.95, 95% CI = (-1.12, -0.79)] to small [emotion: g = -0.37, 95% CI = (-0.63, -0.11)]. Moderation analyses revealed nonsignificant contributions of age, sex, educational attainment, and parental level of education to outcomes. These results illustrate that cognitive impairments are diffuse and salient across the lifespan in NF1. Taken together, these results further demonstrate efforts should be made to evaluate and address cognitive morbidity in patients with NF1 in conjunction with existing best practices., (© 2022 Wiley Periodicals LLC.)

Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Prime editing is a recent precision genome editing modality whose versatility offers the prospect for a wide range of applications, including the development of targeted genetic therapies. Yet, an outstanding bottleneck for its optimization and use concerns the difficulty in delivering large prime editing complexes into cells. Here, we demonstrate that packaging prime editing constructs in adenoviral capsids overcomes this constrain resulting in robust genome editing in both transformed and non-transformed human cells with up to 90% efficiencies. Using this cell cycle-independent delivery platform, we found a direct correlation between prime editing activity and cellular replication and disclose that the proportions between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context. Hence, adenovector particles permit the efficacious delivery and testing of prime editing reagents in human cells independently of their transformation and replication statuses. The herein integrated gene delivery and gene editing technologies are expected to aid investigating the potential and limitations of prime editing in numerous experimental settings and, eventually, in ex vivo or in vivo therapeutic contexts., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: We sought to quantify the proportion of contacts reported by persons with COVID-19 through a short message service (SMS)-linked survey in comparison to the proportion of contacts reported during a follow-up phone-interview. We also sought to assess improvement in contact tracing timeliness associated with sending SMS-linked surveys., Study Design: During December 4-15, 2020, persons identified as COVID-19 cases whose data was entered into Marin County's contact tracing database on even days received a SMS-linked survey and persons whose data was entered on odd days did not; all were called for case investigation and contact tracing. Chi-square test and Fisher's exact test were used to compare demographic data. Chi-square test was used to contrast categorical outcomes, and Wilcoxon's rank-sum test was used for continuous outcomes., Results: Among 350 SMS-linked survey recipients, 85 (24%) responded and 4 (1%) reported contacts using the survey; an additional 303 contacts were reported during phone interviews. Without phone interviews, 99% of reported contacts would have been missed. There was no meaningful difference between study arms in the proportion of contacts notified within 48 h., Conclusions: This SMS-linked survey had low participation and was not useful for identifying contacts. Phone interviews remained crucial for COVID-19 contact tracing., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier Ltd on behalf of The Royal Society for Public Health.)

Background: ModraDoc006 is an oral formulation of docetaxel, which is co-administered with the cytochrome P450 3A4 and P-glycoprotein inhibitor ritonavir (r): ModraDoc006/r. Weekly treatment with ModraDoc006/r had been evaluated in phase I trials in patients with different types of advanced solid tumors, but up to this point in time not in patients with metastatic castration-resistant prostate cancer (mCRPC)., Aim: We assessed safety and pharmacokinetics (PK) of ModraDoc006/r to establish the recommended phase 2 dose (RP2D) in patients with mCRPC., Methods: mCRPC patients, treatment naïve or following abiraterone or enzalutamide treatment, were included. Dose-escalation of ModraDoc006/r was based on safety and docetaxel PK. Antitumor activity was assessed by serum prostate-specific antigen (PSA) and radiological evaluation., Results: Cohort 1 (n = 5) received once weekly ModraDoc006 30 mg with ritonavir 100 mg in the morning, and ModraDoc006 20 mg with ritonavir 100 mg in the evening (30-20/100-100). The mean docetaxel area under the plasma concentration-time curve (mAUC0-inf) was 461 ng/mL × h with 1 dose limiting toxicity (DLT); grade 3 alanine transferase increase. In cohort 2 (n = 6, ModraDoc006/r 30-20/200-200), the mAUC0-inf was 1687 ng/mL × h with 2 DLTs; grade 3 diarrhea and mucositis. In cohort 3A (n = 6, ModraDoc006/r 30-20/200-100), the mAUC0-inf was 1517 ng/mL × h with 1 DLT; grade 3 diarrhea. In cohort 3B (n = 3, ModraDoc006/r 20-20/200-100), the mAUC0-inf was 558 ng/mL × h without DLTs. The mAUC0-inf exceeded estimated exposures of intravenous docetaxel in cohort 2 and 3A, was lower in cohort 1 and was in range in cohort 3B. PSA decreases of >50% occurred in 6/10 evaluable patients throughout the various cohorts. In five radiological evaluable patients, two confirmed partial responses were observed., Conclusion: The RP2D was established at weekly ModraDoc006/r 30-20/200-100. Observed PSA and radiological responses suggest promising clinical activity. These results have led to an ongoing randomized Phase 2b study, comparing weekly ModraDoc006/r with 3-weekly IV docetaxel in patients with mCRPC., (© 2021 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

Background: Based on reassuring short-term foetal and maternal safety data, there is an increasing trend to administer chemotherapy during the second and third trimesters of pregnancy. The pharmacokinetics (PK) of drugs might change as a result of several physiological changes that occur during pregnancy, potentially affecting the efficacy and safety of chemotherapy., Objective: With this analysis, we aimed to quantitatively describe the changes in the PK of docetaxel, paclitaxel, doxorubicin and epirubicin in pregnant women compared with non-pregnant women., Methods: PK data from 9, 20, 22 and 16 pregnant cancer patients from the International Network of Cancer, Infertility and Pregnancy (INCIP) were available for docetaxel, paclitaxel, doxorubicin and epirubicin, respectively. These samples were combined with available PK data from non-pregnant patients. Empirical non-linear mixed-effects models were developed, evaluating fixed pregnancy effects and gestational age as covariates., Results: Overall, 82, 189, 271, and 227 plasma samples were collected from pregnant patients treated with docetaxel, paclitaxel, doxorubicin and epirubicin, respectively. The plasma PK data were adequately described by the respective models for all cytotoxic drugs. Typical increases in central and peripheral volumes of distribution of pregnant women were identified for docetaxel, paclitaxel, doxorubicin and epirubicin. Additionally, docetaxel, doxorubicin and paclitaxel clearance were increased in pregnant patients, resulting in lower exposure in pregnant women compared with non-pregnant patients., Conclusion: Given the interpatient variability, the identified pregnancy-induced changes in PK do not directly warrant dose adjustments for the studied drugs. Nevertheless, these results underscore the need to investigate the efficacy of chemotherapy, when administered during pregnancy.

Importance: Contact tracing is a multistep process to limit SARS-CoV-2 transmission. Gaps in the process result in missed opportunities to prevent COVID-19., Objective: To quantify proportions of cases and their contacts reached by public health authorities and the amount of time needed to reach them and to compare the risk of a positive COVID-19 test result between contacts and the general public during 4-week assessment periods., Design, Setting, and Participants: This cross-sectional study took place at 13 health departments and 1 Indian Health Service Unit in 11 states and 1 tribal nation. Participants included all individuals with laboratory-confirmed COVID-19 and their named contacts. Local COVID-19 surveillance data were used to determine the numbers of persons reported to have laboratory-confirmed COVID-19 who were interviewed and named contacts between June and October 2020., Main Outcomes and Measures: For contacts, the numbers who were identified, notified of their exposure, and agreed to monitoring were calculated. The median time from index case specimen collection to contact notification was calculated, as were numbers of named contacts subsequently notified of their exposure and monitored. The prevalence of a positive SARS-CoV-2 test among named and tested contacts was compared with that jurisdiction's general population during the same 4 weeks., Results: The total number of cases reported was 74 185. Of these, 43 931 (59%) were interviewed, and 24 705 (33%) named any contacts. Among the 74 839 named contacts, 53 314 (71%) were notified of their exposure, and 34 345 (46%) agreed to monitoring. A mean of 0.7 contacts were reached by telephone by public health authorities, and only 0.5 contacts per case were monitored. In general, health departments reporting large case counts during the assessment (≥5000) conducted smaller proportions of case interviews and contact notifications. In 9 locations, the median time from specimen collection to contact notification was 6 days or less. In 6 of 8 locations with population comparison data, positive test prevalence was higher among named contacts than the general population., Conclusions and Relevance: In this cross-sectional study of US local COVID-19 surveillance data, testing named contacts was a high-yield activity for case finding. However, this assessment suggests that contact tracing had suboptimal impact on SARS-CoV-2 transmission, largely because 2 of 3 cases were either not reached for interview or named no contacts when interviewed. These findings are relevant to decisions regarding the allocation of public health resources among the various prevention strategies and for the prioritization of case investigations and contact tracing efforts.

Introduction: ModraDoc006 is a novel docetaxel tablet formulation that is co-administrated with the cytochrome P450 3A4 and P-glycoprotein inhibitor ritonavir (r): ModraDoc006/r., Objectives: This study evaluated the effect of food consumed prior to administration of ModraDoc006/r on the pharmacokinetics of docetaxel and ritonavir., Methods: Patients with advanced solid tumours were enrolled in this randomized crossover study to receive ModraDoc006/r in a fasted state in week 1 and after a standardized high-fat meal in week 2 and vice versa. Pharmacokinetic sampling was conducted until 48 h after both study drug administrations. Docetaxel and ritonavir plasma concentrations were determined using liquid chromatography with tandem mass spectrometry. Safety was evaluated with the Common Terminology Criteria for Adverse Events, version 4.03., Results: In total, 16 patients completed the food-effect study. The geometric mean ratio (GMR) for the docetaxel area under the plasma concentration-time curve (AUC) 0-48 , AUC 0-inf and maximum concentration (C max ) were 1.11 (90% confidence interval [CI] 0.93-1.33), 1.19 (90% CI 1.00-1.41) and 1.07 (90% CI 0.81-1.42) in fed versus fasted conditions, respectively. For the ritonavir C max , the GMR was 0.79 (90% CI 0.69-0.90), whereas the AUC 0-48 and AUC 0-inf were bioequivalent. The most frequent treatment-related toxicities were grade ≤ 2 diarrhoea and fatigue. Hypokalaemia was the only observed treatment-related grade 3 toxicity., Conclusions: The docetaxel and ritonavir exposure were not bioequivalent, as consumption of a high-fat meal prior to administration of ModraDoc006/r resulted in a slightly higher docetaxel exposure and lower ritonavir C max . Since docetaxel exposure is the only clinically relevant parameter in our patient population, the overall conclusion is that combined ModraDoc006 and ritonavir treatment may be slightly affected by concomitant intake of a high-fat meal. In view of the small effect, it is most likely that the intake of a light meal will not affect the systemic exposure to docetaxel. CLINICALTRIALS., Gov Identifier: NCT03147378, date of registration: May 10 2017.

Crizotinib and alectinib are anaplastic lymphoma kinase (ALK)-inhibitors indicated for the treatment of ALK-positive metastatic non-small cell lung cancer (NSCLC). At the currently used fixed doses, interindividual variability in exposure is high. The aim of this study was to investigate whether minimum plasma concentrations (C min ) of crizotinib and alectinib are related to efficacy and toxicity. An observational study was performed, in which ALK-positive NSCLC patients who were treated with crizotinib and alectinib and from whom pharmacokinetic samples were collected in routine care, were included in the study. Exposure-response analyses were explored using previously proposed C min thresholds of 235 ng/mL for crizotinib and 435 ng/mL for alectinib. Forty-eight crizotinib and 52 alectinib patients were included. For crizotinib, median progression-free survival (mPFS) was 5.7 vs. 17.4 months for patients with C min  < 235 ng/mL (48%) and ≥ 235 ng/mL, respectively (P = 0.08). In multivariable analysis, C min  < 235 ng/mL resulted in a hazard ratio (HR) of 1.79 (95% confidence interval (CI), 0.90-3.59, P = 0.100). In a pooled analysis of all crizotinib patients (not only ALK-positive, n = 79), the HR was 2.15 (95% CI, 1.21-3.84, P = 0.009). For alectinib, mPFS was 12.6 months vs. not estimable (95% CI, 19.8-not estimable) for patients with C min  < 435 ng/mL (37%) and ≥ 435 ng/mL, respectively (P = 0.04). Multivariable analysis resulted in an HR of 4.29 (95% CI, 1.33-13.90, P = 0.015). In conclusion, PFS of crizotinib and alectinib treated NSCLC patients is prolonged in patients with C min  ≥ 235 ng/mL and 435 ng/mL, respectively. Therefore, therapeutic drug monitoring should be part of routine clinical management for these agents., (© 2020 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Purpose: We studied EGFR mutations in circulating tumor DNA (ctDNA) and explored their role in predicting the progression-free survival (PFS) of non-small cell lung cancer (NSCLC) patients treated with erlotinib or gefitinib., Methods: The L858R, T790M mutations and exon 19 deletions were quantified in plasma using digital droplet polymerase chain reaction (ddPCR). The dynamics of ctDNA mutations over time and relationships with PFS were explored., Results: In total, 249 plasma samples (1-13 per patient) were available from 68 NSCLC patients. The T790M and L858R or exon 19 deletion were found in the ctDNA of 49 and 56% patients, respectively. The median (range) concentration in these samples were 7.3 (5.1-3688.7), 11.7 (5.1-12,393.3) and 27.9 (5.9-2896.7) copies/mL, respectively. Using local polynomial regression, the number of copies of EGFR mutations per mL increased several months prior to progression on standard response evaluation., Conclusion: This change was more pronounced for the driver mutations than for the resistance mutations. In conclusion, quantification of EGFR mutations in plasma ctDNA was predictive of treatment outcomes in NSCLC patients. In particular, an increase in driver mutation copy number could predict disease progression.

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Capecitabine is an oral pro-drug of 5-fluorouracil. Patients with solid tumours who are treated with capecitabine may develop hand-and-foot syndrome (HFS) as side effect. This might be a result of accumulation of intracellular metabolites. We characterised the pharmacokinetics (PK) of 5-fluorouridine 5'-triphosphate (FUTP) in peripheral blood mononuclear cells (PBMCs) and assessed the relationship between exposure to capecitabine or its metabolites and the development of HFS. Plasma and intracellular capecitabine PK data and ordered categorical HFS data was available. A previously developed model describing the PK of capecitabine and metabolites was extended to describe the intracellular FUTP concentrations. Subsequently, a continuous-time Markov model was developed to describe the development of HFS during treatment with capecitabine. The influences of capecitabine and metabolite concentrations on the development of HFS were evaluated. The PK of intracellular FUTP was described by an one-compartment model with first-order elimination (k e,FUTP was 0.028 h -1 (95% confidence interval 0.022-0.039)) where the FUTP influx rate was proportional to the 5-FU plasma concentrations. The predicted individual intracellular FUTP concentration was identified as a significant predictor for the development and severity of HFS. Simulations demonstrated a clear exposure-response relationship. The intracellular FUTP concentrations were successfully described and a significant relationship between these intracellular concentrations and the development and severity of HFS was identified. This model can be used to simulate future dosing regimens and thereby optimise treatment with capecitabine.

Introduction Oral formulations of docetaxel have successfully been developed as an alternative for intravenous administration. Co-administration with the enzyme inhibitor ritonavir boosts the docetaxel plasma exposure. In dose-escalation trials, the maximum tolerated doses for two different dosing regimens were established and dose-limiting toxicities (DLTs) were recorded. The aim of current analysis was to develop a pharmacokinetic (PK)-toxicodynamic (TOX) model to quantify the relationship between docetaxel plasma exposure and DLTs. Methods A total of 85 patients was included in the current analysis, 18 patients showed a DLT in the four-week observation period. A PK-TOX model was developed and simulations based on the PK-TOX model were performed. Results The final PK-TOX model was characterized by an effect compartment representing the toxic effect of docetaxel, which was linked to the probability of developing a DLT. Simulations of once-weekly, once-daily 60 mg and once-weekly, twice-daily 30 mg followed by 20 mg of oral docetaxel suggested that 14% and 34% of patients, respectively, would have a probability >25% to develop a DLT in a four-week period. Conclusions A PK-TOX model was successfully developed. This model can be used to evaluate the probability of developing a DLT following treatment with oral docetaxel and ritonavir in different dosing regimens.

In recent years new targeted small molecule kinase inhibitors have become available for pediatric patients with cancer. Relationships between drug exposure and treatment response have been established for several of these drugs in adults. Following these exposure-response relationships, pharmacokinetic (PK) target minimum plasma rug concentration at the end of a dosing interval (C min ) values to guide therapeutic drug monitoring (TDM) in adults have been proposed. Despite the fact that variability in PK may be even larger in pediatric patients, TDM is only sparsely applied in pediatric oncology. Based on knowledge of the PK, mechanism of action, molecular driver, and pathophysiology of the disease, we bridge available data on the exposure-efficacy relationship from adults to children and propose target C min values to guide TDM for the pediatric population. Dose adjustments in individual pediatric patients can be based on these targets. Nevertheless, further research should be performed to validate these targets., (© 2020 The Authors Clinical Pharmacology & Therapeutics © 2020 American Society for Clinical Pharmacology and Therapeutics.)

Rodent neocortical neurons undergo prominent postnatal development and maturation. The process is associated with structural and functional maturation of the axon initial segment (AIS), the site of action potential initiation. In this regard, cell size and optimal AIS length are interconnected. In sensory cortices, developmental onset of sensory input and consequent changes in network activity cause phasic AIS plasticity that can also control functional output. In non-sensory cortices, network input driving phasic events should be less prominent. We, therefore, explored the relationship between postnatal functional maturation and AIS maturation in principal neurons of the primary motor cortex layer V (M1LV), a non-sensory area of the rat brain. We hypothesized that a rather continuous process of AIS maturation and elongation would reflect cell growth, accompanied by progressive refinement of functional output properties. We found that, in the first two postnatal weeks, cell growth prompted substantial decline of neuronal input resistance, such that older neurons needed larger input current to reach rheobase and fire action potentials. In the same period, we observed the most prominent AIS elongation and significant maturation of functional output properties. Alternating phases of AIS plasticity did not occur, and changes in functional output properties were largely justified by AIS elongation. From the third postnatal week up to five months of age, cell growth, AIS elongation, and functional output maturation were marginal. Thus, AIS maturation in M1LV is a continuous process that attunes the functional output of pyramidal neurons and associates with early postnatal development to counterbalance increasing electrical leakage due to cell growth.

Background: For oral anticancer drugs, trough concentration (Cmin) is usually used as a target in therapeutic drug monitoring (TDM). Recording of Cmin is highly challenging in outpatients, in whom there is typically a variability in sample collection time after dosing. Various methods are used to estimate Cmin from the collected samples. This simulation study aimed to evaluate the performance of 3 different methods in estimating the Cmin of 4 oral anticancer drugs for which TDM is regularly performed., Methods: Plasma concentrations of abiraterone, dabrafenib, imatinib, and pazopanib at a random time (Ct,sim) and at the end of the dosing interval (Cmin,sim) were simulated from population pharmacokinetic models including 1000 patients, and the values were converted into simulated observed concentrations (Ct,sim,obs and Cmin,sim,obs) by adding a residual error. From Ct, sim,obs, Cmin was predicted (Cmin,pred) by the Bayesian estimation (method 1), taking the ratio of the Ct,sim,obs and typical population concentration and multiplying this ratio with the typical population value of Cmin,sim (method 2), and log-linear extrapolation (method 3). Target attainment was assessed by comparing Cmin,pred with the proposed pharmacokinetic targets related to efficacy and calculating the positive predictive and negative predictive values., Results: The mean relative prediction error and root mean squared relative prediction error results showed that method 3 was out-performed by method 1 and 2. Target attainment was adequately predicted by all 3 methods (the respective positive predictive value of method 1, 2, and 3 was 92.1%, 92.5%, and 93.1% for abiraterone; 87.3%, 86.9%, and 99.1% for dabrafenib; 79.3%, 79.3%, and 75.9% for imatinib; and 72.5%, 73.5%, and 67.6% for pazopanib), indicating that dose adjustments were correctly predicted., Conclusions: Both method 1 and 2 provided accurate and precise individual Cmin,pred values. However, method 2 was easier to implement than method 1 to guide individual dose adjustments in TDM programs.

Olfactory dysfunction is recognized in neurodevelopmental disorders and may serve as an early indicator of global dysfunction. The present meta-analysis measures olfaction effect sizes in attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorders (ASDs), and obsessive-compulsive disorder (OCD). Meta-analysis included 320 ADHD, 346 ASD, and 208 OCD individuals as compared to 910 controls. Olfactory performance deficits were small-to-moderate and heterogeneous (d =  - 0.42, 95% CI =  - 0.59 < δ <  - 0.25). Meta-analytic results indicate that olfactory dysfunction is evident in individuals with ASD and OCD, with small-to-negligible effects in ADHD. These findings imply olfactory dysfunction is related to clinical phenotype in ASD and OCD, but not ADHD, and warrant inclusion in clinical assessment and evaluation of certain neurodevelopmental disorders.

Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo DMD -correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective DMD alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in E1 and E2A . Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% ± 13% of defective DMD alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both DMD gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of β-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of DMD gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of DMD -correcting agents.

Oral administration of docetaxel is an attractive alternative for conventional intravenous (IV) administration. The low bioavailability of docetaxel, however, hinders the application of oral docetaxel in the clinic. The aim of the current study was to develop a population pharmacokinetic (PK) model for docetaxel and ritonavir based on the phase 1 studies and to support drug development of this combination treatment. PK data were collected from 191 patients who received IV docetaxel and different oral docetaxel formulations (drinking solution, ModraDoc001 capsule, and ModraDoc006 tablet) coadministered with ritonavir. A PK model was first developed for ritonavir. Subsequently, a semiphysiological PK model was developed for docetaxel, which incorporated the inhibition of docetaxel metabolism by ritonavir. The uninhibited intrinsic clearance of docetaxel was estimated based on data on IV docetaxel as 1980 L/h (relative standard error, 11%). Ritonavir coadministration extensively inhibited the hepatic metabolism of docetaxel to 9.3%, which resulted in up to 12-fold higher docetaxel plasma concentrations compared to oral docetaxel coadministered without ritonavir. In conclusion, a semiphysiological PK model for docetaxel and ritonavir was successfully developed. Coadministration of ritonavir resulted in increased plasma concentrations of docetaxel after administration of the oral formulations of ModraDoc. Furthermore, the oral ModraDoc formulations showed lower variability in plasma concentrations between and within patients compared to the drinking solution. Comparable exposure could be reached with the oral ModraDoc formulations compared to IV administration., (© 2019, The American College of Clinical Pharmacology.)

Introduction: The pharmacokinetics (PK) of the 20S proteasome inhibitor bortezomib are characterized by a large volume of distribution and a rapid decline in plasma concentrations within the first hour after administration. An increase in exposure was observed in the second week of treatment, which has previously been explained by extensive binding of bortezomib to proteasome in erythrocytes and peripheral tissues. We characterized the nonlinear population PK and pharmacodynamics (PD) of bortezomib in children with acute lymphoblastic leukemia., Methods: Overall, 323 samples from 28 patients were available from a pediatric clinical study investigating bortezomib at an intravenous dose of 1.3 mg/m 2 twice weekly (Dutch Trial Registry number 1881/ITCC021). A semi-physiological PK model for bortezomib was first developed; the PK were linked to the decrease in 20S proteasome activity in the final PK/PD model., Results: The plasma PK data were adequately described using a two-compartment model with linear elimination. Increased concentrations were observed in week 2 compared with week 1, which was described using a Langmuir binding model. The decrease in 20S proteasome activity was best described by a direct effect model with a sigmoidal maximal inhibitory effect, representing the relationship between plasma concentrations and effect. The maximal inhibitory effect was 0.696 pmol AMC/s/mg protein (95% confidence interval 0.664-0.728) after administration., Conclusion: The semi-physiological model adequately described the nonlinear PK and PD of bortezomib in plasma. This model can be used to further optimize dosing of bortezomib.

Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A liquid chromatography-tandem mass spectrometry assay was developed and validated for the nine oral anticancer agents alectinib, cobimetinib, lenvatinib, nintedanib, osimertinib, palbociclib, ribociclib, vismodegib and vorinostat in order to support therapeutic drug monitoring (TDM). The assay was based on reversed-phase chromatography coupled with tandem mass spectrometry operating in the positive ion mode. The assay was validated based on the guidelines on bioanalytical methods by the US Food and Drug Administration and European Medicines Agency. The method was validated over a linear range of 10-200 ng/mL for alectinib, lenvatinib, nintedanib and vismodegib; 50-1000 ng/mL for cobimetinib and palbociclib; 100-2000 ng/mL for osimertinib; 5.00-100 ng/mL for ribociclib; 25-500 ng/mL for vorinostat. Intra-assay and inter-assay bias was within ±20% for all analytes at the lower limit of quantification and within ±15% at remaining concentrations. Stability experiments showed that osimertinib is unstable in the biomatrix and should be shipped on dry-ice and stored at -20 °C until analysis. All other compounds were stable in the biomatrix. The described TDM method was successfully validated and applied for TDM in patients treated with these KIs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A key component allowing a neuron to function properly within its dynamic environment is the axon initial segment (AIS), the site of action potential generation. In visual cortex, AIS of pyramidal neurons undergo periods of activity-dependent structural plasticity during development. However, it remains unknown how AIS morphology is organized during development for downstream cells in the visual pathway (retinal ganglion cells; RGCs) and whether AIS retain the ability to dynamically adjust to changes in network state. Here, we investigated the maturation of AIS in RGCs during mouse retinal development, and tested putative activity-dependent mechanisms by applying visual deprivation with a focus on the AIS-specific cisternal organelle (CO), a presumed Ca 2+ -store. Whole-mount retinae from wildtype and Thy1-GFP transgenic mice were processed for multi-channel immunofluorescence using antibodies against AIS scaffolding proteins ankyrin-G, βIV-spectrin and the CO marker synaptopodin (synpo). Confocal microscopy in combination with morphometrical analysis of AIS length and position as well as synpo cluster size was performed. Data indicated that a subset of RGC AIS contains synpo clusters and that these show significant dynamic regulation in size during development as well as after visual deprivation. Using super resolution microscopy, we addressed the subcellular localization of synpo in RGC axons. Similar to cortical neurons, RGCs show a periodic distribution of AIS scaffolding proteins. A previously reported scaffold-deficient nanodomain correlating with synpo localization is not evident in all RGC AIS. In summary, our work demonstrates a dynamic regulation of both the AIS and synpo in RGCs during retinal development and after visual deprivation, providing first evidence that the AIS and CO in RGCs can undergo structural plasticity in response to changes in network activity.

Gene editing based on homology-directed repair (HDR) depends on donor DNA templates and programmable nucleases, e.g., RNA-guided CRISPR-Cas9 nucleases. However, next to inducing HDR involving the mending of chromosomal double-stranded breaks (DSBs) with donor DNA substrates, programmable nucleases also yield gene disruptions, triggered by competing non-homologous end joining (NHEJ) pathways. It is, therefore, imperative to identify parameters underlying the relationship between these two outcomes in the context of HDR-based gene editing. Here we implemented quantitative cellular systems, based on epigenetically regulated isogenic target sequences and donor DNA of viral, non-viral, and synthetic origins, to investigate gene-editing outcomes resulting from the interaction between different chromatin conformations and donor DNA structures. We report that, despite a significantly higher prevalence of NHEJ-derived events at euchromatin over Krüppel-associated box (KRAB)-impinged heterochromatin, HDR frequencies are instead generally less impacted by these alternative chromatin conformations. Hence, HDR increases in relation to NHEJ when open euchromatic target sequences acquire a closed heterochromatic state, with donor DNA structures determining, to some extent, the degree of this relative increase in HDR events at heterochromatin. Finally, restricting nuclease activity to HDR-permissive G2 and S phases of the cell cycle through a Cas9-Geminin construct yields lower, hence more favorable, NHEJ to HDR ratios, independently of the chromatin structure., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aim: Pharmacokinetics of small molecule kinase inhibitors (KIs) used in cancer treatment may alter with increasing age, but results are conflicting. This study aims to compare exposure to KIs between older and younger patients (≥70 and <70 years) in clinical practice., Methods: KI plasma concentrations of routinely treated patients were measured using validated assays. Calculated trough concentrations were compared in both age groups. For KIs with a clinically meaningful target concentration (erlotinib, imatinib, pazopanib, sunitinib and vemurafenib), influence of older age on target attainment was assessed., Results: We analysed 616 samples from 454 patients (median age: 61; range 20-93 years), treated with dabrafenib (n = 105), erlotinib (n = 49), imatinib (n = 165), pazopanib (n = 63), sunitinib (n = 87), trametinib (n = 95) and vemurafenib (n = 52). Older age did not significantly influence exposure to erlotinib, imatinib, pazopanib, sunitinib, trametinib and vemurafenib. Elderly patients had significantly higher dabrafenib trough concentrations than younger patients (P = 0.02; 62 ng ml -1 (coefficient of variation [CV] 41%), vs. 53 ng ml -1 (CV 46%), respectively). For KIs with a predefined target concentration, 68% of older and 61% of younger patients reached target., Conclusions: In this real-world study, exposure to most included KIs was comparable in older and younger patients, except for dabrafenib, which showed higher exposure in older patients. In the absence of an absolute target for this KI, clinical relevance remains unclear. For all other included KIs, our data suggest no clinically relevant influence of older age on KI exposure., (© 2018 The British Pharmacological Society.)

Purpose: Despite intense research, treatment options for patients with mesothelioma are limited and offer only modest survival advantage. We screened a large panel of compounds in multiple mesothelioma models and correlated sensitivity with a range of molecular features to detect biomarkers of drug response. Experimental design: We utilized a high-throughput chemical inhibitor screen in a panel of 889 cancer cell lines, including both immortalized and primary early-passage mesothelioma lines, alongside comprehensive molecular characterization using Illumina whole-exome sequencing, copy-number analysis and Affymetrix array whole transcriptome profiling. Subsequent validation was done using functional assays such as siRNA silencing and mesothelioma mouse xenograft models. Results: A subgroup of immortalized and primary MPM lines appeared highly sensitive to FGFR inhibition. None of these lines harbored genomic alterations of FGFR family members, but rather BAP1 protein loss was associated with enhanced sensitivity to FGFR inhibition. This was confirmed in an MPM mouse xenograft model and by BAP1 knockdown and overexpression in cell line models. Gene expression analyses revealed an association between BAP1 loss and increased expression of the receptors FGFR1/3 and ligands FGF9/18. BAP1 loss was associated with activation of MAPK signaling. These associations were confirmed in a cohort of MPM patient samples. Conclusions: A subgroup of mesotheliomas cell lines harbor sensitivity to FGFR inhibition. BAP1 protein loss enriches for this subgroup and could serve as a potential biomarker to select patients for FGFR inhibitor treatment. These data identify a clinically relevant MPM subgroup for consideration of FGFR therapeutics in future clinical studies. Clin Cancer Res; 24(1); 84-94. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Introduction: Paediatric dose-finding studies are challenging to perform due to ethical reasons, the limited number of available patients and restricted number of blood samples. In certain cases, the adult pharmacokinetic (PK) exposure can be used as target for dose finding in paediatrics. The aim of this study was to investigate the performance of a paediatric phase I dose-finding clinical trial in silico., Methods: Using an adult pharmacokinetic model, clinical trial simulations were performed to determine the power of a proposed clinical trial design. Power was defined as the fraction of 1000 trials with an area under the plasma concentration-time curve at steady-state (AUC 0-24,SS ) within ±20% of the adult geometric mean AUC 0-24,SS . Different scenarios were compared to optimise the design of the trial. To show the potential of this framework for similar compounds, the current simulation method was also evaluated with adult and paediatric data from literature on sunitinib., Results: At the starting dose of 300 mg/m 2 , the power of the trial design was 66.9%. Power did not improve by dose escalation to 350 mg/m 2 (65.3%). Power increased to 78.9% with inclusion of 10 patients per trial. Paediatric sunitinib PK data were adequately predicted from adult data with a mean prediction error of 1.80%., Conclusion: The performance of PK-based clinical trials in paediatrics can be predicted and optimised through PK modelling and simulation. Application of this approach enables clinical trials in paediatrics to be performed as efficiently as possible while protecting the child from unnecessary harm., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Adequate gentamicin peak concentrations (Cmax) are important for optimal clinical efficacy. Within a critically ill patient, substantial variability in Cmax can occur over time, hampering the usefulness of therapeutic drug monitoring (TDM). The aim of this study was to evaluate the effect of gentamicin dosing based on Cmax after the first dose on gentamicin target attainment in critically ill patients., Methods: From gentamicin-treated critically ill patients, dosing information, clinical parameters, and serum concentrations were collected prospectively. A population pharmacokinetic model was developed using nonlinear mixed-effects modeling to estimate Cmax after each dose. To evaluate the usefulness of routine TDM, percentages of Cmax within (%Cther, 15-20 mg/L), above (>20 mg/L), and below (%Csubther, <15 mg/L) the therapeutic range after the first and second doses were compared. In addition, simulations were performed to evaluate the impact of TDM., Results: Four hundred sixteen measurements from 59 patients receiving 130 gentamicin doses were included. In the 30 patients who received >1 dose, TDM increased %Cther from 40% after a first median dose of 5.0 mg/kg to 50% after the second dose, and decreased %Csubther from 47% to 30%. Simulations using a 5 mg/kg starting dose revealed %Cther after the second dose of 28.4% without and 36.8% with TDM and %Csubther of 56.9% and 29.3%, respectively. Increasing the simulated starting dose to 6 mg/kg increased %Cther after the first dose from 27.7% to 33.5% and decreased %Csubther from 58.6% to 35.6%. TDM after a first dose of 6 mg/kg had no substantial effect on %Cther or %Csubther after the second dose., Conclusions: Gentamicin dosing based on Cmax after the first dose increased %Cther and decreased %Csubther, but did not result in therapeutic Cmax in half of the patients. When simulating a higher starting dose, %Csubther after the first dose decreased, and TDM showed no additional influence. These data suggest that a starting dose of 6 mg/kg should be considered and that repeated Cmax measurements are not of added value.

Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.

Mutations disrupting the reading frame of the ~2.4 Mb dystrophin-encoding DMD gene cause a fatal X-linked muscle-wasting disorder called Duchenne muscular dystrophy (DMD). Genome editing based on paired RNA-guided nucleases (RGNs) from CRISPR/Cas9 systems has been proposed for permanently repairing faulty DMD loci. However, such multiplexing strategies require the development and testing of delivery systems capable of introducing the various gene editing tools into target cells. Here, we investigated the suitability of adenoviral vectors (AdVs) for multiplexed DMD editing by packaging in single vector particles expression units encoding the Streptococcus pyogenes Cas9 nuclease and sequence-specific gRNA pairs. These RGN components were customized to trigger short- and long-range intragenic DMD excisions encompassing reading frame-disrupting exons in patient-derived muscle progenitor cells. By allowing synchronous and stoichiometric expression of the various RGN components, we demonstrate that dual RGN-encoding AdVs can correct over 10% of target DMD alleles, readily leading to the detection of Becker-like dystrophin proteins in unselected muscle cell populations. Moreover, we report that AdV-based gene editing can be tailored for removing mutations located within the over 500-kb major DMD mutational hotspot. Hence, this single DMD editing strategy can in principle tackle a broad spectrum of mutations present in more than 60% of patients with DMD.

Transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases derived from clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems have become ubiquitous genome editing tools. Despite this, the impact that distinct high-order chromatin conformations have on these sequence-specific designer nucleases is, presently, ill-defined. The same applies to the relative performance of TALENs and CRISPR/Cas9 nucleases at isogenic target sequences subjected to different epigenetic modifications. Here, to address these gaps in our knowledge, we have implemented quantitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability. By using these systems, we demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences. In addition, this outcome could also be ascertained for S. pyogenes CRISPR/Cas9 complexes harbouring Cas9 variants whose DNA cleaving specificities are superior to that of the wild-type Cas9 protein. Thus, the herein investigated cellular models will serve as valuable functional readouts for screening and assessing the role of chromatin on designer nucleases based on different platforms or with different architectures or compositions., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Malignant pleural mesothelioma is an aggressive fatal malignancy with a prognosis that has not significantly improved in the last decades. This review summarizes the current state of treatment and the various attempts that are made to improve overall survival for patients with malignant pleural mesothelioma. It also discusses technologies and protocols to test new and hopefully more effective compounds in a more individualized manner. These developments are expected to improve the prognosis for this group of patients.

Background: Immunologic response to a complete vaccine regimen of currently licensed alum-adjuvanted hepatitis B vaccines is reduced in several subpopulations, including older adults, men, obese persons, and smokers. Two phase 3 trials in healthy adults demonstrated that 2 doses over 1 month of an investigational hepatitis B vaccine (HBsAg-1018) induced superior seroprotection rates (SPRs) to 3 doses over 6 months of the licensed vaccine Engerix-B (HBsAg-Eng)., Methods: An exploratory analysis of immunogenicity was conducted in subpopulations from pooled data for the 2 phase 3 trials., Results: In each subpopulation, the peak SPR in the HBsAg-1018 group was statistically significantly higher than the peak SPR in the HBsAg-Eng group. Peak HBsAg-1018 SPRs ranged from 91.6% to 99.7%, while peak HBsAg-Eng SPRs ranged from 67.7% to 92.9%., Conclusion: In these exploratory analyses, 2 doses of HBsAg-1018 induced statistically significantly higher rates of seroprotection than 3 doses of HBsAg-Eng across all subpopulations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Many patients with chronic kidney disease (CKD) are hyporesponsive to currently licensed alum-adjuvanted hepatitis B vaccines, including Engerix-B(®) (HBsAg-Eng). Seroprotection rates (SPRs) are further reduced in CKD patients with diabetes mellitus. Three doses of an investigational hepatitis B vaccine (HBsAg-1018) that uses a Toll-like receptor 9 agonist demonstrated superior SPRs to 4 double doses of HBsAg-Eng in a large phase 3 trial of CKD patients., Methods: A prespecified subgroup analysis of immunogenicity was conducted in CKD participants with type 2 diabetes in the phase 3 trial., Results: In 328 participants, the peak SPR in the HBsAg-1018 group met criteria for noninferiority and superiority to the peak SPR in the HBsAg-Eng group. The peak geometric mean concentration of antibody against hepatitis B surface antigen in the HBsAg-1018 group was statistically significantly higher than in the HBsAg-Eng group., Conclusion: HBsAg-1018 induced significantly higher seroprotection than HBsAg-Eng in CKD patients with diabetes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.