Simultaneous inhibition of DNA-PK and PolÏ´ improves integration efficiency and precision of genome editing.

Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (PolÏ´). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.

A Combined Human in Silico and CRISPR/Cas9-Mediated in Vivo Zebrafish Based Approach to Provide Phenotypic Data for Supporting Early Target Validation.

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts as part of the process for validating potential new drug targets. Traditional approaches (e.g., genetically-modified mice) are optimal for assessing small numbers of genes, but less practical when multiple genes are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation, by providing more confidence in target relevance and facilitating gene selection for definitive loss of function studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including changes in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports further investigation of the potential roles of API5, HSPB7, and LMO2 in human cardiovascular disease. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing.

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods.

Intensive Care Weaning (iCareWean) protocol on weaning from mechanical ventilation: a single-blinded multicentre randomised control trial comparing an open-loop decision support system and routine care, in the general intensive care unit.

INTRODUCTION: Automated systems for ventilator management to date have been either fully heuristic rule-based systems or based on a combination of simple physiological models and rules. These have been shown to reduce the duration of mechanical ventilation in simple to wean patients. At present, there are no published studies that evaluate the effect of systems that use detailed physiological descriptions of the individual patient.The BEACON Caresystem is a model-based decision support system that uses mathematical models of patients' physiology in combination with models of clinical preferences to provide advice on appropriate ventilator settings. An individual physiological description may be particularly advantageous in selecting the appropriate therapy for a complex, heterogeneous, intensive care unit (ICU) patient population. METHODS AND ANALYSIS: Intenive Care weaning (iCareWean) is a single-blinded, multicentre, prospective randomised control trial evaluating management of mechanical ventilation as directed by the BEACON Caresystem compared with that of current care, in the general intensive care setting. The trial will enrol 274 participants across multiple London National Health Service ICUs. The trial will use a primary outcome of duration of mechanical ventilation until successful extubation. ETHICS AND DISSEMINATION: Safety oversight will be under the direction of an independent committee of the study sponsor. Study approval was obtained from the regional ethics committee of the Health Research Authority (HRA), (Research Ethic Committee (REC) reference: 17/LO/0887. Integrated Research Application System (IRAS) reference: 226610. Results will be disseminated through international critical care conference/symposium and publication in peer-reviewed journal. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov under NCT03249623. This research is registered with the National Institute for Health Research under CPMS ID: 34831.

Impact of a five-dimensional framework on R&D productivity at AstraZeneca.

In 2011, AstraZeneca embarked on a major revision of its research and development (R&D) strategy with the aim of improving R&D productivity, which was below industry averages in 2005-2010. A cornerstone of the revised strategy was to focus decision-making on five technical determinants (the right target, right tissue, right safety, right patient and right commercial potential). In this article, we describe the progress made using this '5R framework' in the hope that our experience could be useful to other companies tackling R&D productivity issues. We focus on the evolution of our approach to target validation, hit and lead optimization, pharmacokinetic/pharmacodynamic modelling and drug safety testing, which have helped improve the quality of candidate drug nomination, as well as the development of the right culture, where 'truth seeking' is encouraged by more rigorous and quantitative decision-making. We also discuss where the approach has failed and the lessons learned. Overall, the continued evolution and application of the 5R framework are beginning to have an impact, with success rates from candidate drug nomination to phase III completion improving from 4% in 2005-2010 to 19% in 2012-2016.

Towards a hit for every target.

Technological advances coupled with novel collaborative strategies for compound sourcing and management are poised to transform the utility of high-throughput screening.

Screening ß-arrestin recruitment for the identification of natural ligands for orphan G-protein-coupled receptors.

A variety of G-protein-coupled receptor (GPCR) screening technologies have successfully partnered a number of GPCRs with their cognate ligands. GPCR-mediated ß-arrestin recruitment is now recognized as a distinct intracellular signaling pathway, and ligand-receptor interactions may show a bias toward ß-arrestin over classical GPCR signaling pathways. We hypothesized that the failure to identify native ligands for the remaining orphan GPCRs may be a consequence of biased ß-arrestin signaling. To investigate this, we assembled 10 500 candidate ligands and screened 82 GPCRs using PathHunter ß-arrestin recruitment technology. High-quality screening assays were validated by the inclusion of liganded receptors and the detection and confirmation of these established ligand-receptor pairings. We describe a candidate endogenous orphan GPCR ligand and a number of novel surrogate ligands. However, for the majority of orphan receptors studied, measurement of ß-arrestin recruitment did not lead to the identification of cognate ligands from our screening sets. ß-Arrestin recruitment represents a robust GPCR screening technology, and ligand-biased signaling is emerging as a therapeutically exploitable feature of GPCR biology. The identification of cognate ligands for the orphan GPCRs and the extent to which receptors may exist to preferentially signal through ß-arrestin in response to their native ligand remain to be determined.

The BlueScreen-384 assay as an indicator of genotoxic hazard potential in early-stage drug discovery.

High-throughput cell-based techniques that permit early detection of compound-induced genotoxic damage have recently become available. Methods based on induction of the GADD45a promoter are attractive because multiple intracellular mechanisms that detect genetic damage intersect at this checkpoint gene. Consequently, assays such as GreenScreen HC, which uses p53-competant human TK6 lymphoblastoid cells and a GADD45a-GFP reporter, have been developed. GreenScreen HC allows weekly testing of dozens of compounds using 96-well microplates, with high interassay consistency. BlueScreen HC is a recent advancement, coupling GADD45a to Gaussia luciferase, with several advantages over GADD45a-GFP including the potential for miniaturization. Here we describe implementation of a 384-well BlueScreen assay. For drug discovery programs carrying out iterative analogue synthesis around a chemical lead series, these assays permit assessment of compound genotoxic potential in parallel to, rather than subsequent to, determination of activity at a therapeutic target. We demonstrate comparability of BlueScreen-384 to GreenScreen HC and illustrate the use of BlueScreen-384 to explore the structure-activity relationship around a genotoxic lead molecule to identify nongenotoxic analogues. BlueScreen-384 can reduce the need for costly and time-consuming analogue testing in more traditional genotoxicity tests, such as the Ames test.