Realising the genome's potential in the context of the living cell. Spring 13
While it is true that this bounty of genetic sequence information holds the key to answering many of these questions and to countless future biological and medical discoveries, the past 10 years have clearly demonstrated that the link between the human genome sequence and how genes, genetic variation and genetic control systems drive physiology and cell behaviour is not a simple one.
It is, in fact, highly complex. That complexity has slowed the pace of discovery, delayed the emergence of the new drug targets and therapies anticipated relatively soon after completion of the HGP and tempered the initial wave of excitement.
We have learned, however, that the genome sequence is only the tip of the iceberg and the starting point for the coming revolution in molecular medicine, the emergence of cell- and genebased therapies and the realisation of truly precise treatment strategies. Leveraging the information obtained from the HGP requires understanding of what the genome sequence – and the variations and mutations discovered as the number of genomes sequenced continues to expand – mean in the appropriate biological and physiological context. That context is the living cell. Without correlating genotype to phenotype one cannot begin to understand how changes in nucleotide sequence, epigenetic modifications, structural alterations in chromatin and regulatory control mechanisms can affect cell behaviour, morphology and viability, and impact how a cell interacts with its surroundings.
The next critical phase of the broader ‘omic’ revolution that is driving drug discovery and development lies in developing a precise and complete picture of cell function – both normal and abnormal. Only then will it be possible to explore more fully the role of the genome, the epigenome, the metabolome and the other ‘omes’ that drive cell behaviour. The ultimate value of all of this information will only be attained through its integration and interpretation in the context of human cells. By studying and developing accurate models of human liver cells, for example, grown and maintained in surroundings that approximate their natural environments, it becomes possible to screen drug candidates in preclinical testing and achieve truly predictive toxicology. Studying and modelling neurons will enable mapping of neural networks to pinpoint the etiology of neurodegenerative diseases and identify new drug targets to intervene earlier in disease processes. The development of cardiomyocytes that can be propagated in large-scale cell culture will foster advances in regenerative therapies and improved cell-based assays to assess cardiotoxicity. Research aimed at characterising stem cells and identifying the signals and stimuli that trigger and direct their growth, differentiation and death, will accelerate progress in developing stem-cell derived cell lines for research, preclinical studies and high throughput screening (HTS), and for advancing cell-based strategies to repair and replace damaged or diseased tissues and organs.
Embracing the complexity of the cell
DNA is often described as the fundamental building block of life, but while it is an essential component of any unicellular organism or cell, a strand of DNA cannot on its own multiply or carry out the instructions for life encoded in its chemical subunits. The basic unit of life, therefore, is the cell. It embodies the DNA and ensures that its messages are transcribed and translated. Understanding those functions and characterising them at the molecular level will open new doors for drug discovery, leading to novel drug targets and therapeutic strategies, safer drugs with less chance for adverse side-effects and late-stage clinical failure due to toxicity, and more targeted treatments with well-defined mechanisms of action that will usher in an era of personalised medicine.
The biopharmaceutical industry continues to be plagued by the failure, during large-scale clinical testing or even withdrawal post-marketing, of promising new drugs due to toxic effects, despite extensive preclinical studies and successful Phase I and II clinical trials. The economic toll of these failures is enormous. The loss may surpass $1 billion for a compound that was discovered in an HTS campaign, developed through an intensive optimisation workflow and subject to an array of preclinical analyses and extensive early-stage clinical scrutiny.
Importantly, new and potentially life-saving drugs are being put on the shelf despite years of development and investment (that could have been spent on other promising candidates), because their toxicity could not be anticipated and avoided. Human cell-based models that simulate drug and metabolite processing and their effects in the body offer a promising solution to supplement or replace traditional animal studies and assays currently used for preclinical ADME-tox and pharmacokinetic/ pharmacodynamic (PK/PD) testing.
Embracing the complexity of the cell means understanding its intricate signalling networks and integrated biochemical pathways, comprehending the full range of cell behaviours and appreciating the role a cell plays within a tissue or organism depending on its type, location and environmental cues; then using this knowledge to gain value from a cell-based application, whether in the form of a live-cell assay for high content screening, a cell-based model for ADME-tox testing, or a cell therapy to treat disease. All of these are realistic and valuable goals that can be achieved by applying advanced technologies and methods for growing, processing, imaging and analysing cells and by furthering the industrialisation and standardisation of cell production for therapeutic applications.
The era of live cell imaging
Innovative high-speed, high-resolution imaging technologies that include super-resolution microscopy and a host of related techniques will dramatically enhance the ability of scientists to study cell structure and behaviour at the molecular level. Some super-resolution microscopy technologies, which can achieve 100-80nm spatial resolution, are now enabling live cell imaging and opening a window into the mechanistic workings of intracellular processes. This, in turn is helping enable discoveries and insights not previously possible. The combination of nanometer-scale resolution, high speed signal detection (so that ‘realtime’ imaging means capturing cellular and subcellular processes as they are occurring), and the capability to record, store and analyse the large amounts of data generated is yielding a wealth of new knowledge.
Live cell imaging will help cell biologists overcome one of the main challenges they have faced: fluorescence microscopy has largely been performed in fixed tissue samples and scientists have had to draw conclusions about what drives cell behaviour based on these findings. Other advances in imaging technology, such as electron microscopy, have given researchers a more detailed view of the cell, and each of these offers its own advantages and limitations. Electron microscopy, for example, provides a snapshot of the cell at one point in time and not a dynamic view. No single tool can provide all the answers a cell biologist needs, but together the range of imaging technologies and techniques available offers a window into the cellular world.
Because advanced cellular imaging technologies such as super-resolution microscopy can give researchers the ability to follow a sequence of events over time under changing conditions, they make it possible to take advantage of innovative label-free technologies to observe cell activity without disruptive interventions, and to experiment with nanoparticle-based approaches to probe the intracellular space without disturbing normal cellular function. Advances enabling nanoscale cellular imaging are making it possible to leverage emerging innovations in the field of nanotechnology and apply them to explore highspeed, molecular-scale events. For example, researchers can mimic or inhibit interactions that may impact the integrity or activity of subcellular complexes or organelles.
Latest super-resolution imaging systems can follow labelled proteins within a living cell over time in three-dimensional (3D) space at near molecular resolution. This allows researchers to observe and measure biological activity in live cells growing in a monolayer on a glass slide or cultured in multicellular 3D constructs that more closely imitate human tissues. The images obtained may reveal the translocation of a tagged protein (eg, indicative of G-protein coupled receptor signalling across a cell membrane) or the opening and closing of an ion channel. They may capture the timelapse sequence of protein-protein, receptor-ligand or pathogen-host interactions, for example; depict the precise morphologic and structural changes that take place in a cell in preparation for cell division, apoptosis, or metastasis; reveal the changes underlying stem cell differentiation or pathogenic transformation such as tumorigenesis; or expose the dynamics of engineered nanoparticles in intraand intercellular environments.
The potential for high resolution live cell imaging to accelerate the discovery and characterisation of new drug targets, drug classes and therapeutic compounds is enormous. Consider, for example, if it were possible to study the dynamic movement of individual protein components of microtubules, which are part of the skeleton-like network that maintains cell structure and play an essential role in chromosome alignment during cell division. A drug discovery campaign aimed at identifying compounds capable of disrupting a cancer cell’s ability to replicate by targeting its microtubule assembly could benefit greatly from this information. Looking ahead, the ability to screen and compare compounds in a high throughput assay format by evaluating their ability to inhibit the function of these proteins, thereby disrupting microtubule formation, would yield direct information on the mechanism of action of the compounds. It would no longer be necessary to rely solely on binding assays, on surrogate markers of drug activity, or merely on assumptions.
Not only would there be visual evidence of a compound’s mode of action and interaction with the designated target, but the effects of subsequent lead optimisation efforts could also be chronicled through a visual record. Furthermore, all measurements, data and analyses derived from cellular imaging and mined from data stores could be linked to the images from which they were derived, creating a reliable and easily accessible audit trail and resource for comparative or historical studies. Data of interest would link directly to the relevant stored image.
Similarly, the development of new classes of antibacterial or antiviral drugs would benefit from high-resolution imaging data in live cells that could reveal in greater detail than was previously possible how a pathogen infects a host cell, replicates, or causes cell death. The ability to pinpoint a specific area of vulnerability in a microbe or a key step in the process of infectivity or replication could lead to more effective antimicrobial strategies. Furthermore, identifying drug targets that are highly conserved across microbial species offers the potential for broader therapeutic efficacy.
The power of super-resolution imaging – real-world examples
To illustrate the potential benefits of high-speed, super resolution and high resolution microscopy, below are summarised two published examples of how the technique has been used to gain a clearer understanding of cellular mechanisms. The first study shows how an immune cell can distinguish between free virus and virus-infected cells in the bloodstream. The second example shows how imaging has led to new insights in the conserved spatial organisation of proteins in the ‘divisome’ of bacteria and how that relates to the cytokinetic processes essential for cell division. 3D-Structured Illumination Microscopy (3D-SIM) is a super resolution technique that approximately doubles the resolution in all three dimensions compared to conventional fluorescence-based optical microscopy methods. The result is an eight-times improvement in volume resolution. SR-SIM can achieve lateral resolution of 50-60nm and axial resolution ranging from 150-300nm.
Brown et al1 used 3D super-resolution microscopy to image primary human natural killer (NK) cells. NK cells are components of the human immune system capable of secreting cytotoxic granules that can lyse and kill virus-infected or otherwise pathologically-transformed cells that they recognise as foreign. NK cells are part of the body’s cell-mediated immune defence system; they also secrete chemical signalling compounds called cytokines (eg, interferon) to communicate with other cells.
In the study, images of NK cells after stimulation of different cell surface receptors showed the intracellular accumulation of lytic granules and interferon- gamma at actin mesh synaptic sites. The images demonstrated that while the NK cells can recognise free virus in blood (and specifically influenza particles), this recognition alone did not lead to receptor activation and opening of the actin mesh at the synapses unless there was co-ligation of leukocyte function-associated antigen-1 (LFA- 1). LFA-1 is an integrin that mediates cell adhesion. These findings, and the ability to visualise the remodelling of the synaptic actin using super-resolution imaging, led the authors to propose a novel mechanism to explain the results. They suggested that the NK cells rely on integrin recognition to differentiate between free pathogen and pathogeninfected cells in blood and proposed integrin recognition as a mediator of receptor activation.
In the second example, Strauss et al2 described the application of 3D-SIM super-resolution microscopy to study the dynamic localisation of the FtsZ protein in two types of bacteria undergoing cell division: the rod-shaped Bacillus subtilis and the spherical Staphylococcus aureus. FtsZ is a tubulin-like cytoskeletal protein that polymerises to form the Z ring, which acts as a scaffold during bacterial cell division, recruits other proteins needed for cell division and generates a contractile force through constriction of the Z ring that is required for cytokinesis. Super-resolution 3D-SIM was used to image the architecture of Z ring structures present in the two types of bacteria.
Conventional fluorescence microscopy using green fluorescent protein (GFP) fusion proteins had previously determined that the Z ring is assembled from FtsZ precursors and is a dynamic structure that undergoes continuous subunit turnover. Conventional microscopy also led to the conclusion that the Z ring is ‘a continuous structure of uniform density’. In contrast, SR-SIM revealed a heterogeneous distribution of fluorescently labelled FtsZ throughout the Z ring. Furthermore, gaps in the fluorescent signal suggested a discontinuous structure. The gaps were approximately 118-200nm in size and could not be resolved using conventional fluorescence microscopy. “Moreover, visualisation of these gaps requires that the 3D-SIM image is rotated around the z-axis and viewed in the axial plane in B. subtilis,” stated the authors.
Based on these findings, the authors concluded that FtsZ molecules, as well as other components of the bacterial divisome examined as part of the study, maintain a dynamic bead-like distribution across the Z ring that can change before and during constriction of the ring, challenging the existing concept of a homogeneous, continuous structure that wraps around the cell. These new insights were made possible by the ability to view the entire 3D architecture of the Z ring in live bacteria at high resolution over time. They led to novel theories of how the Z ring constricts and what triggers constriction and cytokinesis.
Cells – the vanguard of personalised medicine
The biotherapeutics industry is increasingly focusing on cell-based therapies. Though still in its early days, there are nearly 3,000 cell therapies in development and more than 500 clinical studies underway worldwide. Cell therapy takes advantage of the body’s innate ability to repair itself. The therapeutic potential of cell therapy spans cell, tissue and organ types, and is being explored across a range of diseases including those with large unmet need such as cancer, heart disease and neurodegenerative disorders. The development of cell therapies depends first and foremost on a strong foundation of knowledge about the cells targeted for use in a clinical application. Well-defined metrics and methods are needed to quantify ranges for what represents normal and optimal cell function and growth in a laboratory and commercial setting. These efforts will not only support the development of robust, reproducible and standardised protocols for earlystage development and testing of therapeutic strategies and later-stage scale-up and manufacturing, but will ultimately be critical to meet regulatory requirements and ensure quality control and the safety of all those who come in contact with the cells.
Advanced technologies and workflows designed to study, grow, process, transform and analyse cells must be in place before routine cell therapy can become a reality. Collaborative efforts are underway to develop, optimise and validate these systems and methods and to transition them from the laboratory to the bedside. Karolinska University Hospital (Stockholm, Sweden) – a pioneer in the advancement of cell therapies, with several clinical trials under way, including for the treatment of cancer and neurological and metabolic disorders – has partnered with GE Healthcare to research and identify solutions that will advance and accelerate the implementation of cell therapies as standard treatment options.
Another facet of cell therapy with tremendous potential for saving lives in the short term is cord blood transplantation to treat hematopoietic cancers and diseases such as sickle cell anaemia. More than 14,000 patients each year in the United States alone are diagnosed with a life-threatening blood cancer such as leukaemia, lymphoma or myeloma. These cancers can often be successfully treated with long-term remissions and cures by replacing the patients’ blood-forming stem cells with healthy haematopoietic stem cells from the blood of a matching donor. Unfortunately, finding a donor that is a good match is often difficult. An alternative solution is to transplant haematopoietic stem cells isolated from umbilical cord blood – an abundant source of stem cells that is typically discarded after a baby is born, but can be collected and banked. Cells from umbilical cord blood do not have to match the recipient as closely as would stem cells from a regular donor.
Cord blood banks and available stores of donor cord blood for transplantation are increasing in number worldwide. This approach to cell therapy would benefit from greater standardisation in cell processing and from advances in techniques to enable robust and reproducible expansion of stem cell populations from the banked cord blood samples. The number of stem cells in any given sample of cord blood may be sufficient to treat a child in need of a transplant, but is typically not enough to treat an adult. A joint project undertaken by the University of California, San Francisco and GE Healthcare is applying HTS and automated high content imaging technology to screen 120,000 compounds to identify those best able to stimulate expansion of stem cells isolated from cord blood. The most promising compounds will then be evaluated in studies designed to foster large-scale haematopoietic stem cell production in amounts needed for subsequent clinical trials.
The future presents many challenges from a healthcare perspective, including an ageing population and economic pressures that will demand preventive and regenerative strategies and less costly, safer therapeutic options that offer greater efficacy. Genomics is enabling stratification of patient populations for drug testing and for treatment. Advances in cell imaging and stem cell technology is making it possible to develop more targeted, safer treatments tailored to specific patient populations. And progress in translational research is driving those treatments through clinical development and to the patient’s bedside. For cell therapies, continued improvements in technology and in workflows and automation to standardise and industrialise the supply chain for cell collection, processing, and delivery will make these new treatments more broadly accessible. By embracing the complexity of the cell and the ever-expanding database of ‘omic’ information, safer, more effective, more personalised medicines are on the horizon.
Dr Amr Abid is Head of Global Strategic Growth at GE Healthcare Life Sciences. Before joining GE Healthcare in 2011, Abid held a number of positions over 10 years at Invitrogen (now Life Technologies), finally as EMEA Leader, Cell Systems. Prior to entering the industry, he spent five years in pharmacology research at the University of Nancy, France.
1 Brown et al. Blood 2012:120 (18):3729-3740.
2 Strauss et al. PLoS Biology 2012:10(9):e10011389.