BioScience Journal 6

BioScience BIOSCIENCE JOURNAL AUTUMN 2015

BSc

JOURNAL WINTER 2015

Biosimilars

Cell Therapy

Genome editing Exciting technology moves forward

Immunology

Bionics

BIOSCIENCE JOURNAL AUTUMN 2015

WELCOME z

Welcome

Genome editing opens up great hope for the future Welcome to the latest edition of Bioscience Journal, which once again celebrates some of the astonishing advances being made by scientists around the world.

One of the key subjects we look at in this edition is the rapidly developing field of genome editing, which offers the promise of dramatic advances in the way we understand a whole range of health conditions.

In so many ways, genome editing is breaking new ground, adding to our understanding of the way the human body works and suggesting new treatments that were barely imaginable even twenty years ago.

John Dean

Editor in chief

It really does feel like the dawn of something big and this edition of the magazine takes a look at some of the work under way to develop what promises to be one of the most significant, if not the most significant, area of research in the history of medical science.

Inevitably with such a groundbreaking science, there are ethical as well as scientific issues to address, and the way forward will undoubtedly prove to be a question of balance.

Exciting though the field is, science cannot ignore those questions. Nor does it seek to. Striking that balance is nothing new to scientists, who live with tough questions every day and the signs are that there is a real willingness to address the concerns surrounding genome editing in order to create a way forward.

That way forward depends on several things, not least of which is investment and it is heartening to see big business and academia pledging resources to develop this exciting science.

At the end of the day, big business has to see commercial gain in any science and it is clear that genome editing is already proving its worth.

Of course, there will be setbacks, blind alleys, false promise, no scientific field is immune

3

from that, but the signs are that the good days will far outweigh the bad for those working on genome editing.

Too early to talk about cures? In common with the scientific community, Bioscience Journal regards the word ‘cure’ with a dose of scepticism. We all know that truly eradicating disease is not that easy, just look at the way ebola turns out to have a sting in the tail long after the sufferer thinks that the virus has been banished and the way supposedly eradicated illnesses pop up in unexpected places.

But so fundamental is genome editing that, if it continues to develop at this pace, it will become more and more difficult to avoid using the word.

BIOSCIENCE JOURNAL WINTER 2015

CONTENTS

BioScience JOURNAL

6

15

20

8

17

28

10

19

44

4

BIOSCIENCE JOURNAL AUTUMN 2015

CONTENTS z

Contents

3

Introduction/Foreword

4-5

Contents

6-13

UK News

14-19

World News

20-27 Biosimilars 28-43 Genome Editing

48

56

44-50 Cell Therapy 52-55 Immunology 56-57 Inhaler Technology 58-62 Bionics

Editor

John Dean john.dean@distinctivepublishing.co.uk

58

52

Design

Distinctive Publishing, Unit 6b, Floor B, Milburn House, Dean Street, Newcastle Upon Tyne NE1 1LE Tel: 0845 884 2385 www.distinctivepublishing.co.uk

Contributors

John Dean & Francis Griss john.dean@distinctivepublishing.co.uk

Advertising

Distinctive Publishing, Unit 6b, Floor B, Milburn House, Dean Street, Newcastle Upon Tyne NE1 1LE Tel: 0845 884 2343 email: john.neilson@distinctivegroup.co.uk www.distinctivepublishing.co.uk

55

62 5

Distinctive Publishing or BioScience Journal cannot be held responsible for any inaccuracies that may occur, individual products or services advertised or late entries. No part of this publication may be reproduced or scanned without prior written permission of the publishers and BioScience Journal.

BIOSCIENCE JOURNAL WINTER 2015

z NEWS

Breakthrough in fight against skin disease Scientists from the University of Surrey have made an important breakthrough in the fight against the flesh-eating tropical skin disease Buruli ulcer.

The team has discovered that the bacteria causes a blood clot in patients’ skin, similar to those that cause deep vein thrombosis (DVT).

The new findings mean that, like DVT, the clots may respond to anticoagulant medicines and heal more quickly with fewer side effects than with antibiotics alone.

Team members hope that the discovery will accelerate the development of a cure for this chronic debilitating disease which affects poor communities in West Africa and can lead to permanent disfigurement and disability. The World Health Organization considers Buruli ulcer to be an emerging threat to public health.

Lead author of the study, Dr Rachel Simmonds from the University of Surrey, said: “This is a huge breakthrough in our understanding of the disease.

“Buruli ulcer is an emerging tropical disease, which is caused by infection with Mycobacterium ulcerans, an organism which belongs to the family of bacteria that causes tuberculosis and leprosy. Around 5,000 cases are recorded each year, the majority in poor rural communities in West Africa, Australia and Southeast Asia where the infection is

thought to occur when people bath in slow running water.

“While antibiotics are currently used to treat Buruli ulcer, they take a long time to work and few people with the disease can afford to pay for extended stays in hospital. The ulcers are often painless, and as a result, early signs of infection are ignored, or thought to be a ‘curse’.

Infected people, often children, are treated by traditional healers rather than modern medicine.

“We hope our research will now enable better treatment combinations that will reduce the lifetime deformity patients have to bear.”

In a separate piece of work, researchers from the University of Surrey and Lund University in Sweden investigated how frequent, longdistance travel is represented in mass and social media in a way that can ignore the health effects.

They found that the images portrayed do not take into account the damaging side effects of frequent travel such as jet-lag, deep vein thrombosis, radiation exposure, stress, loneliness and distance from community and family networks.

6

Lead author Dr Scott Cohen, from the University of Surrey, said: “A man in a sharp suit, reclining in a leather chair, laptop open in front of him, a smiley stewardess serving a scotch and soda. This is often the image of travel, particularly business travel portrayed in TV ads and glossy magazines. But there is a dark side to this ‘glamorised’ hypermobile lifestyle that the media, and society ignores., “The level of physiological, physical and societal stress that frequent travels places upon individuals has potentially serious and long-term negative effects that range from the breaking down of family relationships, to changes in our genes due to lack of sleep.

“It is not only traditional media that perpetuates this image. Social media encourages competition between travellers to ‘check-in’ and share content from far-flung destinations. The reality is that most people who are required to engage in frequent travel suffer high levels of stress, loneliness and long-term health problems. There are also wider implications for the environment and sustainability. In this context, hypermobility seems far from glamourous.”

BIOSCIENCE JOURNAL WINTER 2015

NEWS z

Enzyme research

Scientists at the University of York were part of a research team which found that a recently-discovered family of enzymes can degrade resistant forms of starch. Earlier research established that the enzymes – lytic polysaccharide monooxygenases (LPMOs) – are able to degrade hard-to-digest biomass into its constituent sugars. Now they have been shown to have the ability to ‘chip away’ at other intractable materials such as resistant forms of starch.

New treatment

Collaboration targets degeneration in MS University of Edinburgh scientists are set to work with leading biotechnology business Genzyme, a Sanofi company, to carry out drug discovery research that could reduce neuron damage in the brain.

The collaboration – brokered by Edinburgh BioQuarter’s Business Development team - will focus on identifying therapeutic candidates capable of promoting remyelination and reducing neurodegeneration, mostly in relation to Multiple Sclerosis (MS).

MS is caused by damage to myelin, the protective layer that surrounds nerve fibres.

This damage affects the transmission of electrical signals from the brain to the rest of the body and results in symptoms such as problems with muscle movement, balance and vision.

Over time, MS, patients accrue disability, which usually slowly gets worse - this is related to neurodegeneration.

A natural process called ‘remyelination’ can

repair damaged myelin and restore nerve function.

In MS, however, remyelination is inefficient, something which has captured the interest of the research team.

A new drug, which is being tested, is allowing some patients with terminal diagnosis to maintain a good standard of life. Rucaparib is a type of drug known as a PARP inhibitor and was initially developed in a collaboration between Newcastle University and Cancer Research UK after Rucaparib was derived from research by Roger Griffin and Bernard Golding in the School of Chemistry.

Old remedy

A one thousand year old Anglo-Saxon remedy for eye infections which originates from a manuscript in the British Library has been found to kill the modern-day superbug MRSA in a research collaboration at The University of Nottingham.

Scientists from the University of Edinburgh have discovered a physiologically-occurring molecule that prevents the cells needed to help repair damaged myelin from reaching the area of damage, which limits remyelination.

Coinvestigators Dr Anna Williams, of the MRC Centre for Regenerative Medicine, and Dr Scott Webster hope to identify inhibitors of this molecule or its receptor to prevent the block.

Dr. Williams said: “If successful, this will be a step-change in MS treatment as current treatments are unable to repair the damaged neurons that cause the symptoms of MS. “Ultimately this could reduce neurodegeneration in MS and the accumulation of disability in patients. This treatment could also be used in other diseases where myelin is damaged, such as spinal cord injury.”

Dr. Johanne Kaplan, Vice President of Neuroimmunology Research at Genzyme, stated: “Remyelination-promoting therapies remain an unmet need and would be of great benefit to MS patients.”

7

Dr Christina Lee, an Anglo-Saxon expert from the School of English has enlisted the help of microbiologists from University’s Centre for Biomolecular Sciences to recreate a 10th century potion for eye infections from Bald’s Leechbook an Old English leatherbound volume in the British Library, to see if it works as an antibacterial remedy.

BIOSCIENCE JOURNAL WINTER 2015

z NEWS

‘Chemical harpoons’ could tackle antibiotic problem The global threat of widespread bacterial resistance to antibiotics is one of the greatest challenges facing science and medicine.

Now, the discovery of bacterial ‘chemical harpoons’ could pave the way for a new approach to treating bacterial infections by “disarming” bacteria instead of trying to kill them with antibiotics.

The chemical harpoons, say the team, can be compared to a superglue, whereas all previously known bacterial binding mechanisms can be likened to weak adhesives that require large contact areas for strong binding.

Research led by the University of St Andrews and the John Innes Centre reveals how Streptococcus pyogenes, the cause of many infections including the life-threatening “flesheating disease”, use chemical harpoons to attach themselves to the body.

This tactic is shared by many other bacteria that infect humans, such as Streptococcus pneumoniae, the most common cause of pneumonia in adults, and Clostridium difficile, notorious for causing severe gut infections in hospitalised patients.

The study was funded by the Medical Research

Council (MRC) and led by Dr Uli Schwarz-Linek, structural biologist of the Biomedical Sciences Research Complex at St Andrews, and Dr Mark Banfield, John Innes Centre in Norwich, in collaboration with Professor Manfred Rohde (Helmholtz Center for Infection Research, Braunschweig, Germany).

Dr Schwarz-Linek said: “Among the weaponry of bacteria are protein molecules within hairlike structures displayed on their surfaces,” “These serve the important purpose of allowing bacteria to cling to host tissues, such as the cells lining the lung or the gut. We have discovered how bacteria use surface proteins to achieve this important step in infections using a surprising and particularly efficient method.

“I believe these findings may significantly change our view of how bacteria colonise their hosts. Our discoveries open an avenue for the development of molecules that can deactivate the chemical harpoons and, therefore, prevent bacteria from gaining a foothold in the body.

This is of great interest since it concerns a

8

topic of the highest possible relevance for our society - the fight against bacterial infections.”

Dr Mark Banfield from the John Innes Centre, who co-led the study, said: “It has been very exciting to build on our initial discovery of the unusual bond these bacteria make with their host and to now appreciate how that bond works – all enabled through international collaborative research. Further, using the powerful X-rays available at Diamond Light Source, the UK’s synchrotron facility, we were able to visualise this bacteria/host interaction at the atomic level.”

Dr Des Walsh, Head of Infections and Immunity at the Medical Research Council, said: “Before we can develop new ways of fighting antimicrobial resistance, we need to fully understand how bacteria survive. It is exciting that MRC-funded researchers have discovered a unique insight into how bacteria invade and seize healthy tissue. We recently awarded an additional £5m towards AMR research, and we will continue to support the best, collaborative research that explores new and promising ways to solve the challenge.”

BIOSCIENCE JOURNAL AUTUMN 2015

NEWS z

Scan change

Medical Research Council scientists have developed a new approach to speed up MRI scans for those who cannot hold their breath. Patients need to lie still and, in some cases, hold their breath for several seconds which for many can be difficult.

The new MRI technique exploits the fact that MRI images can be mathematically compressed in a similar way to digital photographs stored in JPEG files so shortening the time taken for the scan

Horses and humans ‘share facial expressions’ Horses share some surprisingly similar facial expressions to humans and chimps, according to new University of Sussex research.

Researchers have shown that, like humans, horses use muscles underlying various facial features - including their nostrils, lips and eyes - to alter their facial expressions in a variety of social situations.

The findings suggest evolutionary parallels in different species in how the face is used for communication.

The study builds on previous research showing that cues from the face are important for horses to communicate on the basis of underlying muscle movement.

The Equine Facial Action Coding System, devised by the Sussex team in collaboration with researchers at the University of

Portsmouth and Duquesne University, identified 17 facial movements in horses. This compares with 27 in humans, 13 in chimps and 16 in dogs.

The study’s co-lead author, doctoral researcher Jennifer Wathan, said: “Horses are predominantly visual animals, with eyesight that’s better than domestic cats and dogs, yet their use of facial expressions has been largely overlooked. What surprised us was the rich repertoire of complex facial movements in horses, and how many of them are similar to humans.

“Despite the differences in face structure between horses and humans, we were able to identify some similar expressions in relation to movements of the lips and eyes. What we’ll now be looking at is how these expressions relate to emotional states.”

The researchers analysed video footage of a wide range of naturally occurring horse behaviours to identify all the different movements it is possible for horses to make with their face.

9

New centre

The University of Manchester is to lead a new £2.9 million molecular pathology research project to improve diagnosis and treatment of non-cancerous diseases within the NHS. The new Manchester Molecular Pathology Innovation Centre will employ and train scientists in the use of emerging techniques which examine molecules within organs, tissues or bodily fluids for markers that assist in diagnosis and treatment.

Company sold

A company doing genetic engineering research to help control pests has been sold to a US-based biotechnology company for $160m. Oxitec uses genetic engineering to control insect pests that spread disease and damage crops, and was founded in 2002 as a spinout from Oxford University. Intrexon Corporation intends to use Oxitec’s existing initiatives to combat diseases like dengue fever as well as agricultural pests.

BIOSCIENCE JOURNAL WINTER 2015

z NEWS

New procedure offers hope for those suffering from blindness

Red grapes could help combat cancer Resveratrol, a chemical found in red grapes, is more effective in smaller doses at preventing bowel cancer in mice than high doses, according to new research.

Previous research looked at high doses of purified resveratrol to study its potential to prevent cancer. It was the first study to look at the effects of a lower daily dose - equivalent to the amount of resveratrol found in one large glass of red wine – comparing it with a dose 200 times higher.

Results from bowel cancer-prone mice given the smaller dose showed a 50 per cent reduction in tumour size while the high dose showed a 25 per cent reduction.

Lower doses of resveratrol were twice as effective as the higher dose in stopping tumours growing, although this effect was only seen in animals fed a high-fat diet.

Samples of tumours from bowel cancer patients given different doses of resveratrol showed that even lower doses can get into cancer cells and potentially affect processes involved in tumour growth.

Resveratrol is a naturally-occurring chemical found in grape skins and other plants.

Laboratory studies have suggested that it may have anti-cancer properties, although results from human trials have been mixed.

However, the researchers stress that the study doesn’t mean drinking red wine reduces cancer risk, as drinking alcohol increase the chances of developing the disease.

Karen Brown, professor of translational cancer research at the University of Leicester, said: “For the first time, we’re seeing that less resveratrol is more. This study shows that low amounts may be better at preventing tumours than taking a high dose.

“The same might be true for other plantderived chemicals and vitamins that are also being studied for cancer prevention. There should be more research looking at the effects of low doses. But this is early laboratory research and the next stage is for clinical trials to confirm whether resveratrol has the same effects in people at high risk of bowel cancer.”

Dr Julie Sharp, Cancer Research UK’s head of health information, said: “This research doesn’t mean that having a glass of red wine will reduce your risk of cancer because you can’t separate the resveratrol from the alcohol, and the increase in cancer risk linked to alcohol outweighs any possible benefits of the resveratrol.

“It’s a fascinating study but we need much more research to understand all the pros and cons of someone taking resveratrol to prevent bowel cancer. However, we do know that keeping a healthy weight along with a balanced diet low in red and processed meat with lots of fibre, including fruit and vegetables, can stack the odds in your favour to lower your risk of developing the disease.”

10

A trial of a new treatment derived from stem cells for people with ‘wet’ age-related macular degeneration (AMD) has started at Moorfields Eye Hospital in London, following a successful operation on a patient.

The operation is a milestone in the London Project to Cure Blindness, which was established ten years ago with the aim of curing vision loss in patients with wet AMD and involves the hospital, the UCL Institute of Ophthalmology, the National Institute for Health Research and Pfizer Inc. The trial is investigating the safety and efficacy of transplanting eye cells derived from stem cells to treat people with sudden severe visual loss from wet AMD. The cells are used to replace those at the back of the eye that are diseased in AMD, using a specially engineered patch inserted behind the retina.

With the first surgery having been successfully performed, the team hopes to determine her initial visual recovery by early December.

Retinal surgeon Professor Lyndon Da Cruz, from Moorfields Eye Hospital, who is performing the operations and is co-leading the London Project, said: “There is real potential that people with wet age-related macular degeneration will benefit in the future from transplantation of these cells.”

The trial will recruit ten patients over 18 months. Each patient will be followed for a year to assess the safety, effectiveness and stability of the cells.

Professor Pete Coffey, of the UCL Institute of Ophthalmology, who is also co-leading the London Project, said: “Although we recognise this clinical trial focuses on a small group of AMD patients who have experienced sudden severe visual loss, we hope that many patients may benefit in the future.”

Dr Berkeley Phillips, UK Medical Director, Pfizer Ltd, said: “Stem cell-derived therapy was only a theory until recent years and to be part of a project that is applying the latest scientific breakthroughs to help restore patients’ eyesight is truly rewarding.”

BIOSCIENCE JOURNAL AUTUMN 2015

NEWS z

Welcome for packaging

The Association of the British Pharmaceutical Industry (ABPI) has welcomed the Government’s proposal to publish the cost of medicines to the NHS on packs of those costing more than £20. Alison Clough, ABPI Acting CEO and Executive Director – Commercial UK, said: “We broadly welcome a scheme to ensure that patients and the public are more aware and informed of the cost of health care in the UK.”

Collaboration showcases research

Stroke research programme under way Treating stroke patients with antibiotics and paracetamol could save up to 25,000 lives each year, researchers at the University of Edinburgh have said.

A £4m study is to investigate whether routinely offering these drugs to stroke patients helps to prevent the complications such as infection and fever.

Researchers say that up to half of stroke patients suffer high temperatures following their illness and a third contract infections that increase their risk of death and disability.

A quarter of patients have difficulty swallowing and face an increased risk of choking as a result.

The initiative will involve almost 4,000 patients from across Europe. It will test whether offering drugs immediately following a stroke reduces the risk of these complications occurring.

Patients aged 66 years or older will be randomly allocated to receive either preventative treatment in the first four days of their hospitalisation, or to standard care.

Those receiving preventative care will be offered paracetamol to prevent high fevers and antibiotics to lower the risk of infections.

Prof Malcolm MacLeod, Professor of Neurology and Translational Neuroscience, said: “We have made great progress in treating stroke, but it still remains a major cause of death and disability.

“This new trial aims to understand how to use existing treatments most effectively and has the potential to reduce risk of death or disability for as many as 25,000 people each year, at very low costs.”

Stroke is the second leading cause of death globally and accounts for the loss of almost seven million lives each year. It is also the second most common cause of long-term disability.

The trial is led by the University of Utrecht and funded by the Horizon 2020 programme.

11

A collaboration involving Queen’s University in Belfast has developed a blood analyser used in hospitals and war zones, The OPTI blood analyser, which was developed by Queen’s Professor AP de Silva, in collaboration with Optimedical Inc and AVL BioScience Corporation, Roswell GA, produces blood test results in less than 30 seconds, enabling rapid medical responses to be carried out. It is being used worldwide in GP surgeries, veterinary surgeries and hospital critical care units.

Industry is on the up

Britain’s £50 billion chemical and pharmaceutical industry continues to show solid growth in 2015. In the latest survey of its members by the Chemical Industries Association most companies predict they will increase sales in the next 12 months. Added to this, almost 40% of businesses will increase capital expenditure.

BIOSCIENCE JOURNAL WINTER 2015

z NEWS

Aspirin could reduce cancer risk Research has shown that a regular dose of aspirin reduces the long-term risk of cancer in those who are overweight.

The findings came out of an international study of people with a family history of the disease conducted by researchers at Newcastle University and the University of Leeds.

“This research adds to the growing body of evidence which links an increased inflammatory process to an increased risk of cancer.

The trial was overseen by Newcastle Hospitals NHS Foundation Trust and funded by the UK Medical Research Council, Cancer Research UK, the European Union and Bayer Pharma.

They found that being overweight more than doubles the risk of bowel cancer in people with Lynch Syndrome, an inherited genetic disorder which affects genes responsible for detecting and repairing damage in DNA. About half of those people develop cancer, mainly in the bowel and womb.

“Obesity increases the inflammatory response.

One explanation for our findings is that the aspirin may be supressing that inflammation which opens up new avenues of research into the cause of cancer.”

Professor John Mathers, Professor of Human Nutrition at Newcastle University who led part of the study, said: “The lesson for all of us is that everyone should try to maintain a healthy weight and for those already obese the best thing is to lose weight. However, for many patients this can be very difficult so a simple aspirin may be able to help this group.”

However, over the course of a ten year study the team found that the risk could be counteracted by taking a regular dose of aspirin.

The collaboration was led by Sir John Burn, professor of Clinical Genetics at Newcastle University and honorary consultant clinical geneticist at Newcastle Hospitals NHS Foundation Trust, who said: “This is important for people with Lynch Syndrome but affects the rest of us, too. Lots of people struggle with their weight and this suggests the extra cancer risk can be cancelled by taking an aspirin.

The trial is part of the CAPP 2 study involving scientists and clinicians from more than 43 centres in 16 countries which followed nearly 1,000 patients with Lynch Syndrome, in some cases for more than ten 10 years.

A total of 937 people began either taking two aspirins every day for two years or a placebo.

When they were followed up ten years later, 55 had developed bowel cancers and those who were obese were more than twice as likely to develop this cancer. Following up on patients who were taking two aspirins a day revealed that their risk was the same whether they were obese or not.

12

Professor Burn said that anyone thinking of taking aspirin on a regular basis should consult their doctor first because aspirin is known to bring with it a risk of stomach complaints including ulcers.

The international team are now preparing a large-scale follow-up trial and want to recruit 3,000 people across the world to test the effect of different doses.

BIOSCIENCE JOURNAL WINTER 2015

NEWS z

Stem cell research shows promise Experts at The University of Nottingham have discovered the first fully synthetic substrate with potential to grow billions of stem cells.

Their research could pave the way for the creation of ‘stem cell factories’, the mass production of human embryonic (pluripotent) stem cells.

The £2.3m research project, led by Morgan Alexander, Professor of Biomedical Surfaces in the School of Pharmacy, and Chris Denning, Professor of Stem Cell Biology in the School of Medicine and funded by the Engineering and Physical Sciences Research Council (EPSRC), could lead to products for use in the treatment of heart, liver and brain conditions.

Professor Alexander said: “The possibilities for regenerative medicine are still being researched in the form of clinical trials. What we are doing here is paving the way for the manufacture of stem cells in large numbers when those therapies are proved to be safe and effective.”

Professor Denning, whose field is in cardiac stem cell research, said: “The field of regenerative medicine has snowballed in the last five years and over the coming five years a lot more patients will be receiving stem cell treatments.

“Clinical trials are still in the very early stages.

However, with this kind of product, if we can get it commercialised and validated by the regulators it could be helping patients in two to three years.”

Conditions of the heart, liver and brain are all under investigation as possible new stem cell treatments. People are already receiving stem cells derived eye cells for eye disorders.

The field of regenerative medicine has snowballed in the last five years and over the coming five years a lot more patients will be receiving stem cell treatments.

Professor Denning

Professor of Stem Cell Biology in the School of Medicine

Blocking escape route offers hope to cancer patients A new drug that blocks cancer’s escape route from chemotherapy could be used to treat lung and pancreatic cancers.

Scientists in human cancer cells and in mice have shown that the drug boosts the effectiveness of conventional chemotherapy.

The drug, discovered at the Institute of Cancer Research in London, is now scheduled to begin its first clinical trials in people patients with lung and pancreatic cancers, both of which have low survival rates.

Most chemotherapies work by damaging the DNA of rapidly dividing cells. In response, cancer cells activate a molecule called CHK1

13

which delays cell division and gives cancer cells time to repair their damaged DNA. The research showed that the new drug could be very effective in blocking CHK1, which could stop cancer cells from repairing DNA damage and prevent them from becoming resistant to the effects of chemotherapy.

Among organisations supporting the work were the the University of Kent, Newcastle University and drug discovery company Sareum. Funding came from Cancer Research UK.

BIOSCIENCE JOURNAL WINTER 2015

z WORLD NEWS

Fly research casts new light on degenerative conditions Researchers at the Babraham Institute and University of Massachusetts Medical School in the United States have developed a new model to study motor neuron degeneration.

The work has already been used to identify three genes involved in the neurodegeneration process.

The model permits genuine ageing studies to take place as changes in neurons can be observed in flies of different ages.

According to the team, the findings could have relevance for understanding the progression of amyotrophic lateral sclerosis (ALS) and other forms of motor neuron disease (MND). ALS is the most common form of adult-onset motor neuron disease and kills more than 1,200 people a year in the UK alone.

The adult fruit fly can live for more than two months in the lab. Furthermore, the fly provides the benefits of rapid development (ten days from egg to adult), allowing highthroughput genetic screens.

The researchers developed a new model to study neurodegeneration in the common fruit fly (Drosophila melanogaster). In contrast to other methods used to study neurodegeneration such as looking at changes in eye morphology or studying larval stages, the researchers focused their attention on studying the neurons in a fruit fly’s leg.

Using the leg allows the detailed study of a single motor neuron, the nerve cell involved in passing signals from the brain to a muscle.

The researchers used the new model to study the role of a protein central to the development of ALS called TDP-43.

Exposing flies to a mutagen and looking for reduced neurodegeneration in their offspring allowed researchers to identify three genes implicated in mediating the effects of TDP-43.

One, shaggy/GSK3, was already known to be associated with the neurodegeneration process but two of them, hat-trickand xmas-2, were new discoveries.

Dr Jemeen Sreedharan, Senior Research Fellow in the signalling research programme at the

14

Babraham Institute and lead author on the paper which the team wrote on their findings, said: “We’re extremely excited about our new approach to using the power of Drosophila genetics.

“Never before has anyone been able to study adult neurodegeneration in an invertebrate system with such ease.

“By modelling the earliest stages of ALS, synaptic and axon degeneration, we have identified three intriguing genetic suppressors of degeneration in the fly and are now validating these results in mammalian neuronal cultures. We hope that by using the fly we can accelerate progress towards eventually developing therapies for ALS and other neurodegenerative diseases.”

Funding for the research was provided by the Medical Research Council, a Motor Neurone Disease Association Lady Edith Wolfson Fellowship and the Max Rosenfeld Fund.

BIOSCIENCE JOURNAL AUTUMN 2015

WORLD NEWS z

An all-natural sunscreen derived from algae

Promising options

German firm CellGenix has launched the CellGenixTM PSC Kit for use in stem cell production. The system can be used for expansion of human pluripotent stem cells, like human embryonic stem cells and human induced pluripotent stem cells. Human pluripotent stem cell research is an expanding field that has potential to change the way human diseases are studied and treated.

Firm bought

Boulevard Acquisition Corp has completed the acquisition of AgroFresh, the specialty chemical business of The Dow Chemical Company. AgroFresh Solutions is a global industry leader in providing specialty chemicals enabling growers and packers of fresh produce to preserve and enhance the freshness, quality and value of their goods and is headquartered in Collegeville, Pennsylvania.

Virus research

Scientists are turning to the natural sunscreen of algae – which is also found in fish slime – to make a novel kind of shield against the sun’s rays.

Reporting in the American Chemical Society’s journal, the team report that existing sunblock lotions typically work by either absorbing ultraviolet rays or physically blocking them.

They say that a variety of synthetic and natural compounds can accomplish this but most commercial options have limited efficiency, pose risks to the environment and human health or are not stable.

Vincent Bulone, Susana C. M. Fernandes and colleagues looked to nature for ways of tackling the problem.

The researchers used algae’s natural sunscreen molecules, which can also be found in reef fish mucus and microorganisms, and combined them with chitosan, a biopolymer from crustacean shells. Testing showed their materials were biocompatible, stood up well in heat and light, and absorbed both ultraviolet A and ultraviolet B radiation with high efficiency.

The authors were funded by European Commission Marie Curie Intra-European Fellowship, the KTH Advanced Carbohydrate Materials Consortium (CarboMat), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and the Basque Government Department of Education.

15

Researchers in South Africa are working on vaccine against the deadly tickborne disease known as Congo Fever.

Prof Felicity Burt, from the Department of Medical Microbiology and Virology, at the University of the Free State, has been awarded a research grant by the National Health Laboratory Service (NHLS) to study candidate vaccines for Crimean-Congo haemorrhagic fever (CCHF) virus and other arboviruses.

BIOSCIENCE JOURNAL WINTER 2015

z WORLD NEWS

Scientific heroes are honoured Scientists who developed products that improve health and contain safer materials have been inducted into a scientific “Hall of Fame”.

They become the newest Heroes of Chemistry, an honour bestowed by the American Chemical Society (ACS), the world’s largest scientific society.

ACS president Diane Grob Schmidt, Ph.D. said: “Thanks to their creative genius and the encouragement and backing of their employers, we have new tools and new hope for treating a number of difficult diseases.”

The latest additions were recognised in a ceremony during the Society’s 250th National Meeting & Exposition in Boston.

They were: • Bristol-Myers Squibb: For the discovery of ELIQUIS® (apixaban), a oral anticoagulant therapy used most often in patients with atrial fibrillation for whom the risks of stroke, bleeding and death are significantly lower than the decades long standard of care. Among those contributing to the development of this drug were Donald Pinto of Churchville, Pa.; Michael Orwat of New Hope, Pa; Mimi Quan of Yardley, Pa.; Patrick Lam of Chadds Ford, Pa. (former Bristol-Myers Squibb employee); Robert Knabb of Avondale, Pa.; and Pancras Wong of Plainsboro, N.J. • Celgene Corporation: For the discovery and development of POMALYST® (pomalidomide), a drug that is changing treatment for cancer patients stricken with multiple myeloma.

Providing an oral alternative to intravenous (IV) medicines, POMALYST® improves overall survival and survival for patients who have exhausted standard treatments in earlier lines of the disease. Honorees include George Muller

of Rancho Santa Fe, Calif., and Roger Shen-Chu Chen of Edison, N.J. • Gilead Sciences, Inc.: For the development of HARVONI®, the first single-tablet regimen for the treatment of chronic hepatitis C, a disease that affects more than three million Americans and 170 million individuals worldwide. HARVONI® contains two direct acting antivirals, sofosbuvir and ledipasvir, and is 94-99 percent effective for the most common form of chronic hepatitis C. HARVONI® was discovered and developed

by Cheng Yong (Chris) Yang of Foster City, Calif.; Bruce Ross of El Granada, Calif.; Michael Sofia of Doylestown, Pa.; John Link and Erik Mogalian of San Francisco, Calif., Benjamin Graetz and Bob Scott of San Mateo, Calif.; and Rowchanak Pakdaman of San Carlos, Calif.

• Pfizer: For the development of XELJANZ® (tofacitinib citrate), a revolutionary oral therapy for the treatment of rheumatoid arthritis, which affects nearly 24 million people worldwide. Honorees include Mark Flanagan of Gales Ferry, Conn.; Michael Munchhof of Salem, Conn.; Paul Changelian of Northville, Mich.; Chakrapani Subramanyam of South Glastonbury, Conn.; Frank Urban of Old Saybrook, Conn.; Rajappa Vaidyanathan of Bangalore, India; Matt Brown of Stonington, Conn.; William Brissette of Stonington, Conn.; Elizabeth Kudlacz, Eileen Elliott, Douglas Ball, Frank Busch, Robert Dugger, and Sally Gut Ruggeri of Groton, Conn.; Michael Fisher of Oxford, Conn.; and Todd Blumenkopf.

Thanks to their creative genius and the encouragement and backing of their employers, we have new tools and new hope for treating a number of difficult diseases Diane Grob Schmidt, PhD ACS president

16

BIOSCIENCE JOURNAL AUTUMN 2015

WORLD NEWS z

Rice work

Scientists have developed a new, simple way to cook rice that could cut the number of calories absorbed by the body by more than half, potentially reducing obesity rates. Team leader Sudhair A. James, who is at the College of Chemical Sciences, Colombo, Sri Lanka, said: “We discovered that increasing rice resistant starch concentrations was a novel way to approach the problem. If the best rice variety is processed, it might reduce the calories by about 50-60 per cent.”

New fuel

Beer compound could help fend off Alzheimer’s and Parkinson’s diseases Scientists have discovered a potential health benefit of hops.

Writing in the American Chemical Society’s journal, they reported that a compound from hops could protect brain cells from damage and potentially slow the development of disorders such as Alzheimer’s and Parkinson’s diseases.

Jianguo Fang and his colleagues, supported by funding from Lanzhou University and the Natural Science Foundation of Gansu Province, noted mounting evidence that oxidative damage to neuronal cells contributes to the development of diseases that originate in the brain.

If scientists could find a way to guard these cells from this type of damage, they might be able to help prevent or slow down Alzheimer’s disease, Parkinson’s disease and other neurodegenerative conditions.

One compound found in hops, called xanthohumol, has attracted the attention of researchers for its potential benefits, including antioxidation, cardiovascular protection and anticancer properties.

The team decided to test xanthohumol’s effects on brain cells and, in laboratory tests, the researchers found that the compound could protect neuronal cells and potentially help slow the development of brain disorders

Researchers in America have come up with an aviation fuel that reduces greenhouse gas emissions. The breakthrough has come from the Energy Biosciences Institute, a partnership led by the University of California Berkeley, the University of Illinois at Urbana-Champaign, and BP. Alexis Bell, a chemical engineer at Berkeley Lab, said: “Biofuel solutions, such as farnesane, mixed directly with petroleum jet fuel have been tested but offer only modest greenhouse gas reduction benefits. Ours is the first process to generate true dropin aviation biofuels.”

New pesticide

AMVAC Chemical Corporation, a business unit of American Vanguard Corporation, has introduced AUTILUS™ turf fungicide, a new product that can be used to control anthracnose on golf courses.

Anthracnose is destructive to golf course turf, developing as foliar blight or basal rot and has, over the years, developed resistance to a number of fungicides.

Nobel prize for medicine has been awarded This year’s Nobel prize for medicine has gone to Irish-born William Campbell, Satoshi Omura of Japan and Tu Youyou — the first-ever Chinese medicine laureate.

Campbell and Omura were cited for discovering avermectin, derivatives of which have helped lower the incidence of river blindness and lymphatic filariasis, two diseases caused by parasitic worms that affect millions of people in Africa and Asia.

Tu discovered artemisinin, a drug that has helped significantly reduce the mortality rates of malaria patients.

The committee said: “The two discoveries have provided humankind with powerful new means to combat these debilitating diseases that affect hundreds of millions of people annuall. The consequences in terms of improved human health and reduced suffering are immeasurable.”

River blindness is an eye and skin disease that ultimately leads to blindness. About 90 per cent of the disease occurs in Africa, according to the World Health Organization.

Lymphatic filariasis can lead to swelling of the limbs and genitals, called elephantiasis, and

17

it’s primarily a threat in Africa and Asia. The WHO says 120 million people are infected with the disease, with about 40 million disfigured and incapacitated.

William Campbell is a research fellow emeritus at Drew University in Madison, New Jersey, in the United States. Satoshi is a professor emeritus at Kitasato University in Japan and is from the central prefecture of Yamanashi. Tu is chief professor at the China Academy of Traditional Chinese Medicine.

BIOSCIENCE JOURNAL WINTER 2015

z WORLD NEWS

Firms announce expansions

Two American bioscience companies have announced expansions and new jobs.

Seventh Wave, a consulting-based contract research organisation for the pharmaceutical and medical device industry, is expanding in Missouri.

The company is increasing its presence in St. Louis by building a 50,000 sq. ft. plant, a $11 million investment expected to create 42 jobs over the next six years.

The move has the support of the Missouri Department of Economic Development, which says it reaffirms the state’s importance to the industry.

Governor Jay Nixon said: “With more than 50,000 workers employed in over 3,500 bioscience companies that call Missouri

home, the state is a hub for research and innovation.

“Seventh Wave’s decision to continue its growth right here in our state will help bring more discoveries from the laboratory to the market as well as create in-demand jobs in an emerging field.”

Seventh Wave was established in 2003.

With clients around the globe, the company provides services for discovery and preclinical drug development.

Previously operating out of a 15,000 sq.ft facility, the company plans to triple its footprint by constructing a facility which will allow it to dedicate 60 per cent of the space to

offices and laboratories. An additional 20,000 sq. ft. of shell space will be incorporated into the expansion to anticipate future growth.

John Sagartz, President, CEO, and founder of Seventh Wave Laboratories, said: “We are excited about this growth,” This expanded facility will enable us to better serve existing clients, attract new clients, grow existing service lines, and launch new service lines.”

The company plans to hire the 42 new employees in the technical and scientific professions with the new space fully operational sometime in October.

In a separate move, Dallas-based Allied BioScience, which relocated its headquarters to North Texas in 2014, is expanding again.

The firm creates antimicrobial surface coatings and CEO Michael Ruley said: “As we add new clients and technology, we anticipate even more growth in Dallas-Fort Worth, especially within the hospital environment.”

With more than 50,000 workers employed in over 3,500 bioscience companies that call Missouri home, the…state is a hub for research and innovation.

Jay Nixon Governor

18

He said that the company will keep its global headquarters at Crescent Court in Uptown within 100 Crescent Court, while expanding its national operations to Legacy Drive in Plano.

The new Plano office will total 6,000 square feet and hold 25 of the company’s employees.

Allied BioScience teams up with researchers and scientists on American university campuses to help refine and test antimicrobial surface coatings. The company’s flagship technology is called SurfaceWise. The biotech firm also has a number of other products in various stages of research and development.

BIOSCIENCE JOURNAL AUTUMN 2015

WORLD NEWS z

Research grant

Caladrius Biosciences, Inc, a US-based cell therapy company, has received an award from the National Eye Institute of the National Institutes of Health to investigate 3D retinal constructs to restore vision in patients with age-related macular degeneration. The grant of $671,633 will support a threeyear study led by Dr. Hans S. Keirstead, Caladrius’ Senior Vice President, Research, and Chief Science Officer.

Fighting back

Slowing down time A team of chemists and biologists at the Institute of Transformative BioMolecules (ITbM) at Nagoya University in Japan have found new molecules that change the ‘circadian’, or body clock, of mammals.

Most living organisms have a biological clock with a 24-hour rhythm, which regulates body functions such as sleep, hormone secretion, and metabolism.

Disruption of the rhythm by genetic mutations and environmental factors such as jet lag may lead to sleep disorders, as well as obesity, cancer and mental problems.

A team of synthetic chemists, chronobiologists and theoretical chemists have now discovered

the first molecule that targets the clock’s protein CRY. The team says that the study could be useful for developing further molecules that can control the circadian rhythm in mammals, which may overcome diseases and control reproductive activity in animals which would assist in food production.

Takashi Yoshimura, an animal biologist and professor at ITbM, who led the research from a biological perspective., said: “We hope we can make further use of synthetic chemistry to make bioactive molecules that can control the circadian rhythm of animals and gain further insight into the circadian clock mechanism, which will surely contribute to medical applications, food production and advances in clock research.

“This has been a wonderful experience for me to work with chemists and we will continue to work together for more exciting results to come.”

19

The US Government has awarded a contract for the final phase of construction of the National Bio and Agro-Defense Facility in Manhattan, Kansas. The $834 million award will secure completion of the centre, which will research possible threats to the nation’s food supply and agricultural sector.

Learning more

The Cancer Prevention and Research Institute of Texas in America has awarded UT Southwestern Medical Center researchers more than $7.5 million to improve diagnostic and therapeutic services relating to cancers of the brain, breast, throat, and bone, as well as to improve scientific understanding of cancer biology. UT Southwestern received an additional $4 million to recruit emerging cancer scientists.

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

The growth and growth of the

biosimilars market The growth in the global biosimilars market is continuing to accelerate at a remarkable rate, driven by an increasing interest in cheaper versions of major drug brands as health services try to reduce spiralling drug bills.

One of the companies which has highlighted the recent growth is pharmaceutical industry analyst visiongain, which forecasts that the biosimilars market worldwide will be worth $16.03 billion in 2019, a five-fold increase on last year.

The report, entitled Biosimilars and FollowOn Biologics: World Industry and Market Prospects 2015-2025, covers a wide range of treatments, including monoclonal antibodies and fusion proteins, insulins, erythropoietin, interferons, human growth hormone and fertility hormones.

Arshad Ahad, a pharmaceutical industry analyst for visiongain, said: “Biologics are crucial for the treatment of many serious chronic disorders such as cancer, diabetes, hepatitis and autoimmune diseases.

However, those effective but highly complex therapies come at a price, with some treatments costing more than $75,000 a year.

“Biosimilars can offer the benefits of branded biologic therapies but at a lower price, making them more affordable for individual patients, as well as entire nations, in the face of restricted healthcare budgets.

“With patents for many blockbuster biologics set to expire over the next ten years, we predict the biosimilars market will grow rapidly between now and 2025, achieving high revenues.

“However, it will face many challenges, including opposition from originator companies, expensive and lengthy development times and fragmentation of the

market. Nevertheless, as the long-awaited approval of the first biosimilar in the US in 2015 shows, biosimilars are here to stay and their rise to prominence is just beginning.”

A second visiongain report found that the industry generated revenue of an estimated $3.25bn in 2014 alone and that more major companies are becoming involved as they acknowledge the threat to their businesses from the cheaper versions.

According to Pharma Leader Series: Top 25 Biosimilar Drug Manufacturers 2015-2025, developing countries, especially China and India, will see more companies entering the biosimilars industry and becoming bigger national players between now and 2025.

Researchers working for visiongain attribute this to the fact that both countries have an ageing population and a growing demand for cheaper healthcare. They says that the investment, coupled with the growing expertise, could result in Chinese and Indian companies dominating the biosimilars industry.

Bochung Lam, a healthcare industry analyst for visiongain, said: “Biosimilars are no longer a niche concept in the pharmaceutical business. Biosimilars are a reality, and the threat they pose to the revenue of originator pharmaceutical companies is very real.

“At the same time, they also represent a huge opportunity for the same companies that they threaten. This is leading more innovative pharmaceutical companies to develop biosimilars in an attempt to enter that lucrative and growing business.

“Our study notes several big pharma companies already have R&D pipelines for biosimilar production and are in late-stage development or clinical trial phases. They are tackling the threat that biosimilars pose by looking to make it part of their business models. This is also leading to partnerships with biosimilar manufacturers in an attempt to enter the business, as well as acquisitions of biosimilar companies as the large firms look to expand their businesses.” visiongain expects the trend to continue from 2015 to 2025, as the biosimilars market expands and matures, opening up vast revenue potential.

20

Bochung said: “We believe that fast developing medical industry will continue to grow from 2015 to 2025, achieving vast increases in sales. visiongain has high expectations of the biosimilar market and for prominent companies that operate in it.”

A third report from visiongain forecasts that the overall world market for biosimilar monoclonal antibodies (mAbs) alone will exceed $4bn by 2020. Biosimilar Monoclonal Antibodies: World Industry and Market Outlook 2015-2025 predicts that the biosimilar mAbs market will multiply in size from 2015 to 2025.

Alyscia Curtis, a pharmaceutical industry analyst for visiongain, said: “The future of the biosimilar mAbs market looks promising with a number of drugs in the R&D pipeline.

“In 2014, several companies have established presences in the biosimilars mAbs market.

Although that overall market was worth just $114m in 2014, visiongain predicts it to exceed $4bn by 2020, with high further rises.”

Biosimilars can offer the benefits of branded biologic therapies but at a lower price, making them more affordable for individual patients, as well as entire nations, in the face of restricted healthcare budgets.

Arshad Ahad

Pharmaceutical industry analyst for visiongain

BIOSCIENCE JOURNAL WINTER 2015

FEATURE z

21

BIOSCIENCE JOURNAL WINTER 2015

ADVERTORIAL

Advances in analytical characterization of biosimilars The substantial improvement in the power of analytical methods to compare different versions of a given protein molecule should be taken into account when considering the need for clinical studies for designation of biosimilarity. Arguably, demonstration of comparative PK, allied to post-registration monitoring, will provide more discriminatory clinical evidence than large pre-approval therapeutic equivalence studies.

By Dr Frits Lekkerkerker NDA Advisory Board member

Formerly Chair of the Dutch MEB and CHMP member.

Expert in Internal Medicine / Endocrinology / Clinical Pharmacology and Biosimilars Recently, Gerard et al. concluded that extrapolation to other indications of biosimilars on the basis of throughout analytical characterization of biosimilars can be done.1 Their message is that the analytical methods available today have evolved enormously compared to the time the first biosimilars came to the market. On analytical grounds it is possible to predict efficacy and safety. Will this have consequences for the registration requirements in the near future?

In 2006, the CHMP came to a positive opinion for the first biosimilar product Omnitrope (genotropin) after a long approval process.

This procedure initiated the publication of biosimilar EU guidelines. The principle behind these guidelines is that similarity demonstration is crucial. Full demonstration of efficacy and safety is no longer required, but a biosimilar application should be supported by an extensive comparability exercise at quality, preclinical as well as at clinical level. The idea behind this approach is that for biologicals comparability is impossible to demonstrate on analytical grounds alone.

Since 2006, several biosimilars have been approved in the EU. Clinical data were limited; studies were often restricted to one indication, being the most sensitive indication to show a potential difference.

Since 2006, major advances have been made in analytical techniques. Exquisite methods exist nowadays to characterise the primary amino

acid sequence, the tertiary conformational structure, posttranslational modifications (e.g. glycosylation) and to assess any impurities and degradation products both after release and during shelf life. Biological activity is further compared by in vitro and in vivo activity assays.

These advances in technology have already influenced regulators. The recent clinical EU guideline mentioned that extrapolation should be considered in the light of the totality of data, i.e. quality, non-clinical and clinical data.2 Gerard et al highlight the possibilities available today to fully establish analytical comparability with quality tools and receptor assays alone and that these possibilities are able to fully justify extrapolation to other indications. The authors point out that these analytical possibilities are already in place for approved biologicals in case of manufacturing changes. Analytical comparability of the product before and after the manufacturing change is common practice. Only in an exceptional case is a clinical efficacy study performed and seldom is an immunogenicity study initiated.

Extrapolation to all the indications of the reference product is essential to the concept of biosimilarity. It is regrettable that clinicians sometimes have difficulty understanding this extrapolation. It requires a full understanding of all the required analytical, preclinical and clinical study data available. It is the task of regulators to explain and convince the medical

22

community about the background of the approval of biosimilar products, a few have already done so.3 The present abilities to demonstrate on the analytical level similarity/comparability to the reference product raise the question of whether there is still a need for all the clinical data currently required. How much efficacy and safety will really be needed for the approval of biosimilar products in the future? Would it not be possible to restrict the comparability exercise to analytical and just PK/PD data alone? The clinical data currently required are costly and burdensome.

Is this compatible with the correct use of our medical resources? Institutional review boards sometimes hesitate to approve these extensive studies with their major burden on patients.

BIOSCIENCE JOURNAL WINTER 2015

One reason to ask for long-term clinical studies is the risk of unexpected immunogenicity.

However the experience gained during the last 10 years has failed to identify any immunogenicity-related issues for approved biosimilars. In a few cases, quality-related issues were already identified in the preauthorization phase as being potentially relevant for an increased immunogenicity risk. It is possible to identify and control these immunogenicity-related risks for biosimilar candidate product.4 With the advances in analytical technologies, the risk of incremental immunogenicity can, to a large extent, be avoided by analytical characterization, batch release testing and stability testing.

PK/PD studies in volunteers could give valuable information on immunogenicity.

ADVERTORIAL

Pre-registration, short-term immunogenicity testing will also give valuable information. The need for pre-registration clinical data should be decided on a case by case basis depending on what is known about the referent biological and on available analytical and pre-clinical data. Accumulation of additional information on longer-term safety, including immunogenicity, is already a standard element of the EU Risk Management Plan in the post registration phase. Immunogenicity would be much better studied by less costly postauthorization monitoring. These studies will provide a better and more sensitive approach for detecting real world differences in efficacy, safety and immunogenicity and are less costly.

Yes, analytical advances are a challenge for regulators. They make it possible to consider

23

requiring much less expensive, pre-registration clinical study data.

References 1 Gerard TL, Johnston G, Gaugh DR. Biosimilars: Extrapolation of Clinical Use to Other Indications. Generics and Biosimilars Initiative. (GaBI Journal). 2015;4(3):118-24.

2 EMEA/CHMP/BMWP/42832/2005 Rev1 (2014) 3 Position of Finnish Medicines Agency Fimea, Interchangeability of Biosimilars. http://www.fimea. fi/instancedata/prime_product_julkaisu/fimea/ embeds/fimeawwwstructure/29197_Biosimilaarien_ vaihtokelpoisuus_EN.pdf 4 Chamberlain PD. Multidisciplinary approach to evaluating immunogenicity of biosimilars: lessons learnt and open questions based on 10 years’ experience of the European Union regulatory pathway. Biosimilars 2014, 4, 23-43

For more information: info@ndareg.com www.ndareg.com

Guiding you all the way

NDA supported over 38% of the new medicinal products that were approved in Europe in 2014 With 100 experts and a unique Advisory Board covering every therapeutic area, NDA can support small as well as large, multi-national pharmaceutical companies with strategic advice and operational support to get their medicines to market and keep them there.

NDA’s support spans right across a development program, from early development to lifecycle management of a medicinal product, incorporating regulatory affairs, health technology assessment, pharmacovigilance and quality assurance.

NDA - Let’s bring medicines to the world

Regulatory Affairs

Pharmacovigilance

www.ndareg.com | info@ndareg.com

Health Technology Assessment

NDA Advisory Board

NDA Group | Boston | London | Munich | Stockholm | Zurich

Providing the formula for successful recruitment.

www.chemify.co.uk

BIOSCIENCE JOURNAL WINTER 2015

ADVERTORIAL

DEPArray™ Technology unleashes the power of NGS Next-Generation Sequencing (NGS) has the potential to revolutionize clinical oncology by providing direct, actionable molecular information about tumor cells. Such information could be used to stratify patients for appropriate clinical trials assignment, select of efficacious, personalized therapies, and, in routine use, to monitor therapy.

The potential power of NGS, however, is largely neutralized by current sample preparation practices, which allow mixtures composed of tumor cells and normal cells to enter the NGS assay workflow. The background of normal diploid cells in the analyzed tumor sample dilutes signals associated with clinically relevant, quantifiable genetic features, such as copy-number variation and loss of heterozygosity. With an admixture of normal cells present in the sample, the prevalence of a mutation within a tumor cannot be assessed.

Resolving sample heterogeneity prior to NGS analysis would eliminate the ambiguity that precludes productive clinical utilization of NGS data, but conventional methods of cell separation cannot produce the 100% pure preparations of tumor cells required.

Flow-based methods require large cell loads, typically unavailable in real life clinical specimens, and are capable only of producing enriched cell preparations, because flow itself is indiscriminate. Similarly, labor-intensive microdissection methods can yield enriched tumor preparations, but irregular, branching tumor cell infiltration patterns make pure tumor cell separation, isolation, and recovery impossible.

The DEPArray™ system from Silicon Biosystems resolves cellular heterogeneity, enabling NGS-based molecular characterization of pure cell subpopulations.

The system can sort, manipulate, and collect individual cells, or groups of cells, with 100% specificity and purity. Compatible with a variety of clinical oncology samples, such as enriched blood, and fresh, frozen, or formalin-fixed paraffin-embedded (FFPE) tissue, the DEPArray™ system enables separation, isolation, and recovery of pure cell preparations composed of phenotypically distinct cell types.

Any cell that can be identified using positive or negative selection of fluorescent markers or probes can be isolated by the DEPArray™ system. Morphological features, such as size and circularity can be included in the selection criteria. A pure preparation of tumor cells can be recovered from a dissociated cells

prepared from an FFPE tumor biopsy, or fresh or frozen tissue, and a pure preparation of circulating tumor cells (CTCs) can be isolated from an enriched blood sample.

The DEPArray™ platform consists principally of a fluorescence microscope, a digital camera, a single-use microfluidic cartridge, computer hardware, and software. Imagebased fluorescence and brightfield microscopy are combined with dielectrophoresis to enable interrogation and direct manipulation of thousands of individual cells.

Dielectrophoretic forces induced between upper and lower electrodes embedded in the cartridge trap suspended cells into a grid formation. Thus immobilized, the cells can be rapidly interrogated for phenotypic characteristics, such as size, shape, and the presence or absence of one or more target surface proteins. For recovery, cells of interest are mobilized and isolated by stepwise transfer to adjacent cages leading to a separate clean buffer volume in a separate area within the cartridge, and subsequently eluted from there without contamination of other cells.

The DEPArray™ system unleashes the power of NGS for diagnostic assays by enabling preanalytical cell type purification from enriched blood, and fresh, frozen, and FFPE-preserved clinical samples. NGS assay workflow sample input composed of 100% pure tumor cells enables facile resolution of genetic alterations present in assayed cells.

By enabling the addition of precise sample preparation to the NGS assay workflow, the DEPArray™ system brings precision medicine

26

concepts such as personalized therapy, molecular monitoring of response to therapy, and liquid biopsy into the realm of practical possibility. Pre-analytical resolution of tumor heterogeneity is a major step forward for precision medicine.

By Farideh Bischoff

Executive Director of Scientific Affairs and Member of the Board

Gianni Medoro

Chief Technology Officer

Nicolò Manaresi Chief Scientific Officer

ADD PRECISION TO UNDERSTANDING CANCER GENETICS

Resolving Tumor Heterogeneity by Digitally Sorting 100% Pure Cells

DEPArrayTM technology can sort and recover homogeneous pools of phenotypically identical cells from heterogeneous cell suspensions obtained from disaggregated FFPE tissues, allowing precise characterization of quantitative genomic traits from your tissue sections.

w w w. s i l i c o n b i o s y s t e m s . c o m

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

Genome editing Exciting technology moves forward Editing the human genome is a ground-breaking technique which is raising the hopes of millions of people afflicted by inherited disease.

opening up possibilities in many areas of research.

Research has already made significant advances in possible therapies for diseases as diverse as sickle-cell anaemia, cystic fibrosis and HIV.

Although these procedures are becoming more accurate and easier to perform, there are still errors in the process and these are being researched to develop the editing process.

Among other conditions to benefit may be muscular dystrophy and Muscular Dystrophy UK is funding research into therapies including editing out faulty genes to treat the condition Duchenne Muscular Dystrophy.

Genome editing techniques, of which there are three main types, all use enzymes to cut strands of DNA, removing a faulty gene to enable it to be replaced with a correct version.

There is a lot at stake because there is a huge range of medical conditions with a major genetic element where genome editing could provide the answer.

Scientific research is seen as at a critical stage with some approaches already being tested in clinical trials. One of the projects is funded in partnership with the Duchenne Children’s Trust.

For example, one in 200 children in the UK is born with mitochondrial disease inherited from their mother. The mitochondria are cell structures which contain an amount of DNA but which are separate from the cell nucleus.

Such work, similar to that being carried out into other conditions by researchers across the planet, could lead to major advances for patients and families wanting to avoid passing on mutations to a new generation and could also be used to treat people already suffering from serious inherited conditions.

While children inherit DNA in their cell nuclei from both parents, the mitochondria are found in the egg only and are, therefore, inherited only from the mother. Most children with mitochondrial disease will have mild or undiagnosed symptoms but in some cases it can be very serious, even fatal.

Indeed, recent advances in genome editing, particularly the technique known as CRISPRCas9, have made the process much easier,

Recently, scientists at Newcastle University developed a technique of mitochondrial donation where the nucleus of an affected

28

cell is introduced to a cell with healthy mitochondria donated by a third party creating a baby which technically has three genetic parents.

Genetic editing could, in the future, eliminate the need for the donated mitochondria by fixing the error in the mother’s own cells.

Also under way is work on techniques to introduce genetically edited cells to adults suffering from conditions such as sickle-cell anaemia, HIV and cystic fibrosis.

In HIV, the treatment has the potential to stop the virus being transmitted to other immune cells by altering the genes the virus uses as a pathway.

In cystic fibrosis, affected cells in the airway and at other sites could be replaced with a healthy version, relieving symptoms.

These techniques have shown promise in laboratory cultures.

Gang Bao, a bioengineering researcher at the Georgia Institute of Technology, in Atlanta in the United States, has already used CRISPR to correct the sickle-cell mutation in human cells grown in a dish.

Therapies such as these could provide treatment and even a cure for the individual

BIOSCIENCE JOURNAL WINTER 2015

FEATURE z

The group has also called for widespread discussion among scientists, ethicists and the wider public about how these emerging techniques may in future be applied clinically, in human reproductive cells and early embryos, to treat or prevent serious genetic disease.

The Wellcome Trust

CONTINUED ON PAGE 30

29

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

CONTINUED FROM PAGE 29

concerned but, because they do not affect the reproductive cells, they would not be passed on to future generations.

One issue with which researchers are grappling is the number of genes implicated in an individual disease.

Some, such as sickle-cell anaemia, are caused by a single mutation making the path to a treatment straightforward. Others, including diabetes, are more complicated, involving several genes and meaning the road to an effective treatment will be longer.

However, with exciting advances in fields like genome editing come ethical considerations.

Since the beginning of September 2015, there have been two major calls for a debate on the regulatory restrictions placed on the science, in order to allow more research on human embryos.

The first call came from a group of research and funding organisations in the UK, including the Wellcome Trust, who said: “The coalition of research funders and learned societies will continue to fund and support research of this kind, as well as studies that further progress and refine these technologies.

“The group has also called for widespread discussion among scientists, ethicists and the wider public about how these emerging techniques may in future be applied clinically, in human reproductive cells and early embryos, to treat or prevent serious genetic disease.”

The call was followed a week later by a

similar one from the international Hinxton Group, a network of stem cell researchers, bioethicists and experts on policy and scientific publishing, who met in Manchester and also concluded that a debate was needed.

In a consensus statement, the group said that research involving editing the human genome, including research with human embryos, was essential to gain basic understanding of biology and reproductive cells The statement said: “In comparison with earlier techniques, modern genome editing technologies and CRISPR/Cas9 in particular are not only very precise, but also easy, inexpensive and, critically, very efficient.

“In addition, since the last round of debates, other areas of science and medicine have likewise advanced; for example, we can now sequence entire genomes quickly and inexpensively.

“The goal of the Hinxton Group is to inform the ongoing debates and to provide useful guidance to decision-makers regarding use of these technologies in humans, and in particular their use to intervene in the human germline.”

The germline is the cells that allow genetic material to be passed onto the next

30

generation through reproduction and includes sperm and eggs.

For all the ethical considerations, there is general agreement that genome editing offers great potential.

Dr Rob Buckle, Director of Science Programmes at the Medical Research Council, said: “As genome editing technologies evolve, it’s vital that the regulatory framework remains robust and adapts so that the full potential of genome editing can be realised in a scientifically, ethical and legally rigorous way.”

Katherine Littler, Senior Policy Advisor at the Wellcome Trust, said: “As with any emerging technology, the potential for genome editing to be applied as a therapeutic tool in future deserves careful consideration. It’s essential that we start these discussions early, by engaging in an open and inclusive debate involving scientists, ethicists, doctors, regulators, patients and their families, and the wider public.”

Innovation.

Imagine the possibilities.

At Janssen, we have big dreams.

We imagine a time when diseases can be treated before people feel sick. When those in need can access life saving medicines, no matter where they are. And when treatment options are so convenient people can spend more time doing what they love.

It’s about radically challenging the way diseases are thought of, dealt with, prevented and intercepted. Now, and in the future.

By collaborating with the brightest minds in every field, we’re turning big visions into game-changing solutions. Because patients are waiting.

We are Janssen. We collaborate with the world for the health of everyone in it.

Learn more at www.janssen.com

Janssen Research & Development, LLC The image depicted contains models and is being used for illustrative purposes only.

BIOSCIENCE JOURNAL WINTER 2015

ADVERTORIAL

Genome-editing tools offer Pharmaceutical Companies new opportunities for drug discovery and development Advances in genome editing technologies will be another powerful tool available to scientists in Janssen R&D in their efforts to discover and develop safe and effective medicines.

By Inès Royaux, PhD Senior Scientist at Janssen R&D

The ability to engineer precisely targeted modifications within the mammalian genome using meganucleases, zinc-finger nucleases (ZFNs) or transcription activator-like (TAL) effector nucleases (TALENs), has great potential for drug discovery and development. These first and second generation approaches have advanced to the clinic and are currently in early stage human trials. The technologies underlying both ZFNs and TALENs rely on targeting specific genes within the genome using a protein-based recognition ‘cassette’ engineered to recognize and bind to a given genetic sequence. Highly precise gene targeting can be achieved but often requires complex protein engineering and intracellular delivery can be challenging. These limitations have been a deterrent to the wide-scale adoption of the technologies.

much excitement due to its simplicity, high efficiency, high speed, and low cost.

It has been widely acknowledged as a major breakthrough in biology and indeed many researchers including Janssen R&D immediately recognized the potential of this groundbreaking technology. In 2014, Emmanuelle Charpentier and Jennifer Doudna received the Dr. Paul Janssen Award for Biomedical Research in recognition for their discovery of this DNA editing strategy. “The work of Drs. Doudna and Charpentier has the potential to make a significant impact on human health, which is the very heart of Dr. Paul Janssen’s legacy, as well as our mission at Johnson & Johnson.” said Paul Stoffels MD, Chief Scientific Officer of Johnson & Johnson, the parent company of Janssen Pharmaceuticals.

Recently, the discovery of the CRISPR/ Cas9 based genome editing has generated

An attractive aspect of the CRISPR approach is the delivery via a lentiviral format for

The work of Drs. Doudna and Charpentier has the potential to make a significant impact on human health, which is the very heart of Dr. Paul Janssen’s legacy, as well as our mission at Johnson & Johnson.

Paul Stoffels MD

Chief Scientific Officer of Johnson & Johnson, the parent company of Janssen Pharmaceuticals.

32

large-scale loss-of-function and gain-offunction screens, similar to the lentiviral shRNA platform. Through a partnership with the Broad Institute, the Functional Genomics Consortium (FGC), Janssen scientists are using cutting-edge lentiviral CRISPR libraries for a variety of applications including for the identification and validation of new drug targets in complex cellular systems. These include induced pluripotent stem (iPS) cellderived neurons and intestinal organoids where an understanding of mechanism of action or resistance to therapeutics is critical for the discovery of promising new medicines.

Exciting data has already been generated in proof-of-concept studies and ongoing experiments are expected to provide novel insights into the proteins and pathways

BIOSCIENCE JOURNAL AUTUMN 2015

involved in the initiation and progression of diseases of interest to Janssen R&D. While the initial focus within Janssen and other groups has been in the application of gene editing technologies for basic and translational research, a number of biotech and startup companies (many of then spun out from the original ground breaking work in academia) have begun exploring the therapeutic applications of genome editing in humans. Janssen has invested in exploratory studies in the therapeutic space by partnering with Poseida Therapeutics, a spinout of Transposagen, for the development of T-cell therapies based on adoptive transfer of chimeric antigen receptor (CAR) T-cells. In this collaboration, Poseida Therapeutics will deploy Transposagen’s latest technologies to

ADVERTORIAL

reengineer T-cells by inactivating particular receptor genes and then expressing an artificial cell surface receptor to target cancer cells. With this existing collaboration, Janssen is hoping to generate off-the-shelf therapy to treat and potentially cure cancers.

being applied in disease modelling. However we note that while the protocols for executing these studies are well known, it is by no means simple or straight forward to apply them – experience and perseverance are additional critical ingredients!

Given the insights gained from the tremendous growth in the application of gene editing across a wide spectrum of research, it is now possible to choose from the different nuclease-based gene editing technologies the most appropriate tool for a particular application. For example in the case of iPSC engineering (where the goal is introduction of specific point mutations), Janssen has opted for the well validated ZFN-based gene editing tool. A series of isogenic MAPT mutant iPSC lines have already been generated and are

In conclusion, gene editing based on the ZFN, TALEN and CRISPR technologies have rapidly become indispensable tools in pharmaceutical research. They provide a means to rapidly model human disease in a way that was not possible before, and as such provide a transformational advance that will facilitate and catalyze the search for better therapies, as we keep in mind the quote of Dr. Paul Janssen, “the patients are waiting”.

33

BIOSCIENCE JOURNAL WINTER 2015

ADVERTORIAL

MRC Harwell: New opportunities for partnerships The Mary Lyon Centre at MRC Harwell has developed into a centre of excellence for the creation, breeding, archiving and phenotyping of the laboratory mouse. It continues to advance, harnessing new technologies, such as those in genome engineering, to provide a wide range of services and training for mouse genetics research. The Centre now stands as a mature research facility with the capacity to accommodate new partnership programs with both academia and industry.

The Mary Lyon Centre opened in 2004 as a large, purpose-built mouse facility. It is integrated within MRC Harwell, a site with a long history of research in mouse genetics, including the discovery of X-chromosome inactivation by the eminent geneticist Mary Lyon. Today, MRC Harwell conducts innovative, translational research into many aspects of mouse genetics.

The Centre houses approximately 55,000 mice and one of the largest repositories of frozen genetically altered mouse lines in the European Mouse Mutant Archive (EMMA). It is a major partner in the International Mouse Phenotyping Consortium (IMPC).

We offer a comprehensive range of services for using the mouse for biomedical research.

Our portfolio covers services for the creation, breeding, housing and archiving of genetically altered mice. We provide a wide range of specialist phenotyping support, including the analysis of embryonic developmental defects, metabolism, behaviour and sensory assessment, clinical chemistry, haematology and pathology.

In addition to these services, the Centre also offers an extensive portfolio of training opportunities for those who use the mouse as a laboratory animal, including training courses in Home Office modules, cryopreservation of sperm and embryos, and conditional transgenic techniques.

We have gained ISO accreditation by introducing management processes that maximise capacity whilst ensuring a high quality of service delivery. The Centre now stands as a national facility for the use of the laboratory mouse in medical research, with robust processes for quality control.

The Centre has demonstrated its capability through its key contribution to the IMPC, which has the ambitious goal of creating and phenotyping knockout mice lines for every gene in the mouse genome, providing a wealth of functional genomic data and novel mouse models for medical research. Data from this can be freely accessed and mouse lines ordered at mousephenotype.org. A considerable proportion of these lines were produced and phenotyped by the Centre.

34

We are sponsors for the CRACK-IT initiative established by the NC3Rs, and are developing a novel phenotyping paradigm to promote the ethical use of mice in biomedical research in partnership with both academia and industry. CRACK-IT supports the development of new technologies and approaches that will contribute to the reduction and refinement of animals in bioscience research. Our home cage monitoring system, one of the CRACKIT Challenges, could allow behavioural phenotypes to be identified with minimal intervention, by enabling assessment of the activity, behaviour and interaction of mice in the cages they were reared in.

Through these initiatives and others, the Mary Lyon Centre at MRC Harwell has shown itself to be an exceptional facility. It has demonstrated its capacity to accommodate ambitious collaborative programs, and is now looking to establish new partnerships with both academia and industry.

BIOSCIENCE JOURNAL WINTER 2015

ADVERTORIAL

CRISPR/CAS9: It’s a good time for gene editing – don’t ruin your experiments with poor antibodies!

Biomedical researchers have long sought the “magic wand” for targeted genome editing in an attempt to affect phenotypic outcome. A number of methods allow manipulation of gene function, including viral vectors, homologous recombination, RNAi, and nucleasebased gene editing systems. These approaches, however, are costly, time-consuming, and unsuitable for large-scale studies.

The most recent CRISPR/CAS9 gene editing technology uses a naturally occurring bacterial nuclease from Streptococcus pyogenes (Sp). The Cas9 system uses a RNA-guided endonuclease technology which allows for inducing indel mutations, specific sequence replacements or insertions and large deletions or genomic rearrangements at any desired location in the genome. In addition, Cas9 can also be used to mediate up- or downregulation of specific endogenous genes or to alter histone modifications or DNA methylation.

In this system, a single multi-domain Cas9 catalyzes a double-strand break in the target DNA composed of a 20-bp sequence matching a guide RNA (gRNA) protospacer and an adjacent downstream 5’-NGG nucleotide sequence (the protospacer-adjacent motif (PAM)) that can be targeted to precise genomic locations. Since the Cas9 nuclease is targeted to its cognate DNA locus through a simple RNA sequence, CRISPR/Cas9 is more powerful and versatile than other system that uses proteinbased targeting, requiring only RNA redesign to change target specificity.

APPLICATIONS OF CRISPR/CAS9

Three main applications of the S. pyogenes Cas9 nuclease have been described, each which relies on a specific Cas9 protein.

The first relies on the use of a nuclear localized Cas9 protein bearing wild-type activity to induce single double strand break leading either to NHEJ (Non-Homologous End-Joining) or to HDR (Homology Directed Repair).

Without a homologous repair template, NHEJ can result in indel mutations, disrupting the target sequence. Alternatively, targeted

mutations can be made with a homologous repair template and by exploiting the HDR pathway.

The second, a mutated Cas9 protein, Cas9D10A, exhibits only site-specific single-strand nicks and does not activate NHEJ, enabling extra precision in gene targeting. Using a pair of sgRNA-directed Cas9 nucleases, it is possible to induce large deletions or genomic rearrangements, such as inversions or translocations.

The third protein, nuclease-deficient Cas9 or dead Cas9 (dCas9), two single point mutations inactivate Cas9 cleavage activity, but do not alter DNA binding to its cognate DNA target locus. This dCas9 can be used to target various effectors (i.e. transcriptional activators, repressors, fluorescent proteins) in a sequencespecific manner without cleavage. This dCas9 can mediate the up- or downregulation of specific endogenous genes or alter histone modifications or DNA methylation at any genomic location.

Cas9 from other species than S. pyogenes have their own PAM sequence and interesting properties. Cas9 from S. aureus (SaCas9) has similar gene editing efficiency to that of SpCas9, but is 1kb smaller. This is ideal for adeno-associated virus (AAV) delivery, which has a packaging limitation of ~4.5kb. Recently, a newly characterized class II nuclease called Cpf1 promises to deliver simple and precise genome engineering by relying on cutting properties, compactness and ability to rely on just one RNA. Cpf1 interesting features portend a future for genomics in which researchers will have any number of editing tools depending on the material being edited.

36

THE USE OF ANTIBODIES FOR CRISPR/CAS9 STUDIES

A good CRISPR-Cas9 antibody is more than just specific. Attempts to generate codonoptimized Cas9 to increase Cas9 expression efficiency have not been successful. Therefore it is critical to choose highly validated antiCas9 antibodies for downstream applications.

A number of methods now also require more than a simple check of the expression of Cas9 using an untagged antibody. Diagenode has developed the largest and most comprehensive set of anti-SpCas9 antibodies, raised against the N or C-terminus of Cas9 nuclease, to address a range of research needs. Each antibody has been highly validated for very specific applications: • Chromatin immunoprecipitation (CRISPR/ Cas9 polyclonal antibody) • Immunofluorescence, western blot, and immunoprecipitation (extremely specific clone 4G10; clone 7A9) • Experiments with fusion proteins – using an antibody raised against C-terminus of Cas9 protein, validated in IF, WB and IP.

CONCLUSION

CRISPR-Cas9 technology is a powerful tool for cell genome reprogramming and molecular biology research. This genome-editing system gives remarkable new possibilities due to its simplicity, high efficiency, and versatility. High quality anti-Cas9 antibodies are crucial for the detection of the Cas9 protein in the edited experimental system.

BIOSCIENCE JOURNAL AUTUMN 2015

BEST CRISPR/CAS9 ANTIBODIES ON THE MARKET! Diagenode offers a broad range of highly validated CRISPR/Cas9 antibodies, recommended for different applications (WB, IF, IP and chromatin IP). These antibodies have been raised against the N- or C-terminus of the Cas9 nuclease.

% of input

ChIP using the Diagenode Cas9 antibody

FLAG

Cas9, PPI

Cas9, A2508-001

ChIP using CRISPR/Cas9 polyclonal antibody

(C15310258)

Immunofluorescence using CRISPR/Cas9 monoclonal antibody 4G10 (C15200216)

Western blot using CRISPR/Cas9 C-terminal monoclonal antibody (C15200223)

Which CRISPR/Cas9 antibody is the best for your application?

Applications Antibody

Special features

WB

IF

IP

ChIP Antibody raised against

CRISPR/Cas9 polyclonal antibody (C15310258)

For chromatin immunoprecipitation

++

++

++

+++

N-terminus of Cas9 nuclease

CRISPR/Cas9 C-terminal monoclonal antibody (C15200223)

For experiments with fusion proteins

++

++

+

no

C-terminus of Cas9 nuclease

CRISPR/Cas9 monoclonal antibody 4G10 (C15200216)

More sensitive and specific than the clone 7A9 (in WB and IF)

+++ +++

++

no

N-terminus of Cas9 nuclease

CRISPR/Cas9 monoclonal antibody 7A9 (C15200203)

Validated in WB, IF and IP

++

++

++

no

N-terminus of Cas9 nuclease

CRISPR/Cas9 - HRP monoclonal antibody 7A9 (C15200215)

Allows reduction of steps in the WB procedure and to increase of sensitivity

+++

no

no

no

N-terminus of Cas9 nuclease

PROMO

All information concerning our CRISPR/Cas9 antibodies are available at www.diagenode.com ORDER ANY CAS9 ANTIBODY from our catalogue and GET 10% DISCOUNT. Promo code: BIOCAS9, offer valid until January 30, 2016.

www.diagenode.com Please contact us for more information Europe Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (OugrĂŠe) // Belgium // Phone: (+32) 4 364 20 50 // E-mail: info@diagenode.com USA Diagenode Inc. / 400 Morris Avenue, Suite 101 // Denville, NJ 07834 // USA // Phone: (+1) 862 209-4680 // E-mail: info.na@diagenode.com

BIOSCIENCE JOURNAL WINTER 2015

ADVERTORIAL

Characterization of CRISPR/Cas9introduced Mutations using the Guide-it™ Indel Identification Kit Streamlined method for characterizing the variety of indels introduced by genome editing technologies: Ideal for identifying indels introduced by CRISPR/Cas9 genome editing in a cell population A complete kit containing all of the components needed to amplify, clone, and prepare modified target sites for DNA sequence analysis: The Guide-it Indel Identification Kit includes reagents for PCR amplification directly from cells and for ligation-free cloning in 15 minutes The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system is the newest tool for targeted genome editing. This genome editing technique is the most flexible method so far. This system is based on two key components that form a complex: the Cas9 endonuclease and a target-specific RNA (single guide RNA or sgRNA) that guides Cas9 to the genomic DNA target site.

Double strand breaks that are introduced to genomic DNA by Cas9 nuclease can be repaired via the homologous recombination (HR) or nonhomologous end joining (NHEJ) cellular repair mechanisms. HR requires an identical (or nearly identical) DNA template to repair the break, and results in full correction of the DNA cleavage. However, DNA damage is more commonly repaired by NHEJ. In this case, cellular machinery ligates the two broken ends of the DNA without a template donor.

This process is more error prone than HR, and often insertions and deletions (indels) are incorporated, resulting in a DNA sequence that differs from the wild-type sequence.

A STREAMLINED METHOD FOR INDEL IDENTIFICATION

Any cell in a population has the ability to correct endonuclease cleavage by either HR or NHEJ, resulting in a variety of DNA sequences at the target site in different cells. To identify the range of indels at the genomic DNA target site in a cell population after editing, we developed a streamlined workflow to amplify, clone, and analyze target sites by DNA sequencing (Figure 1).

This protocol uses a small sample of the cell population as a template for direct PCR amplification of the genomic region containing the endonuclease target site.

The use of Terra PCR Direct Polymerase allows amplification directly from crude cell lysates without the need for genomic DNA purification (Figure 1, step 1). The resulting PCR pool of DNA fragments, containing different mutations at the target site, are cloned into a pUC19 vector using In-Fusion Cloning, a

Figure 1. Workflow for the Guide-it Indel Identification Kit

ligation-free cloning system (Figure 1, step 2). After introduction into optimized Stellar competent cells, colony PCR is used to amplify the inserts (Figure 1, step 3). Finally, DNA sequencing of the amplified inserts is used to identify indels (Figure 1, step 4).

KNOCKOUT OF ACGFP1 USING CRISPR/CAS9 GENOME EDITING

CRISPR/Cas9 genome editing technology was applied to disrupt a single copy of AcGFP1 integrated in the genome of HT1080 cells (HT1080-AcGFP1). These cells exhibit strong green fluorescence (Figure 2, red trace).

Therefore, AcGFP1 knockout can be assessed by monitoring GFP expression by flow cytometry.

In cells that were co-transfected with two plasmids, one expressing Cas9 and the other expressing an sgRNA targeting AcGFP1, a significant percentage of the population (67%) lost GFP expression, due to mutation of the AcGFP1-encoding gene (Figure 2).

The different indels created at the AcGFP1 locus in the cell population were identified using the Guide-it Indel Identification Kit (according to the protocol in Figure 1). Cells (2x105) from the transfected cell population

38

Figure 2. Flow cytometry analysis of HT1080AcGFP1 cells. Cells were analyzed before (red line) and after (green) transfection with plasmids encoding Cas9 and an AcGFP1-targeting sgRNA. were collected and used directly as a template for PCR amplification of the genomic AcGFP1encoding gene targeted by Cas9/sgRNA. The amplified genomic DNA fragments (Figure 3, lane 1) contain a mixture of the different indels that occurred via NHEJ repair at the AcGFP1 locus following double strand breakage.

The PCR products were cloned into a prelinearized pUC19 vector using In-Fusion Cloning. Eight of the resulting bacterial clones

ADVERTORIAL

Figure 3. PCR product amplified from the Cas9 sgRNA target sequence.

Lane M: Marker; Lane 1: Amplified AcGFP1 target site (* indicates location of PCR product).

Figure 5.

Sequencing results of the AcGFP1 target sequences obtained from eight different clones. expression was lost in approximately 51% of the cell population.

Figure 4. Agarose gel of colony PCR products from eight clones. Lane M: Marker; Lane 1–8: Individual clones.

The Guide-it Indel Identification Kit was used to characterize the nature of the indels created

by Cas9/sgRNA in the cell population. Cells from the same population analyzed in Figure 7, which contained CD81 expressing and nonexpressing cells, were used as template for direct PCR amplification of the targeted CD81 genomic site (Figure 8).

were selected for colony PCR using Terra PCR Direct Polymerase with primers that amplified the insert. The amplified DNA was analyzed on an agarose gel, and all colonies showed a band of the correct size (Figure 4).

The PCR products were sequenced using the provided sequencing primers. The obtained DNA sequence was aligned with the wild-type AcGFP1 sequence (Figure 5). The sequences showed a high rate of an inserted “T” at the Cas9/sgRNA cleavage site in the different clones. One clone had a four nucleotide deletion immediately upstream of the PAM site. Only one clone contained the wild-type AcGFP1 sequence (Figure 5).

DISRUPTION OF CD81 USING CRISPR/ CAS9 GENOME EDITING CD81 is a membrane receptor expressed in most cell types. Using a FITC-tagged antibody against CD81, expression can be monitored by flow cytometry (Figure 6).

The endogenous CD81 gene was targeted for CRISPR/Cas9 genome editing by transfecting HeLa cells with two plasmids encoding Cas9 and a CD81-targeting sgRNA, respectively.

Five days after transfection the cells were harvested, labeled with an anti-CD81-FITC antibody, and analyzed by flow cytometry (Figure 7). The analysis indicated that CD81

Figure 6. Flow cytometry analysis of HeLa cells, labeled with and without an anti-CD81-FITC antibody

Figure 8. PCR products from amplification of the targeted CD81 site from the cell population analyzed in Figure 7. Lane M: Marker; Lane 1: Amplified CD81 target site (* indicates location of PCR product).

Figure 9. Agarose gel of colony PCR products directly from six clones. Lane M: Marker; Lane 1–6: Individual clones.

Figure 7. Flow cytometry analysis of HeLa cells labeled with the anti-CD81-FITC antibody after transfection with Cas9 and sgRNA targeting the CD81 gene.

After cloning the PCR products, six bacterial clones were picked for colony PCR amplification of the CD81 target sequence using Terra PCR Direct Polymerase. The amplified DNA was analyzed on an agarose gel, and all colonies showed a band of the correct size (Figure 9).

obtained sequencing data from the different clones showed a wide variety of deletions and insertions at the targeted CD81 gene, emphasizing the variation in the outcome of NHEJ repair between individual cells.

Takara and the Takara logo are trademarks of TAKARA HOLDINGS, Kyoto, Japan. Clontech, the Clontech logo, and Guide-it are trademarks of Clontech Laboratories, Inc. All other marks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. © 2015 Takara Bio Europe SAS

The amplified PCR products were sequenced using the provided sequencing primer, and the sequencing data were aligned to the wild-type CD81 sequence (Figure 10). The

Figure 10. Sequence analysis of six clones obtained from cells transfected with Cas9/ sgRNA targeting the CD81 gene

39

Tools for Successful Genome Editing G U I D E - I T

™

P R O D U C T S

F O R

C R I S P R / C A S 9

Efficient production, screening, and application of sgRNAs The CRISPR/Cas9 system is revolutionizing genome editing, providing unprecedented targeting potential, efficiency, and simplicity. From experimental design to screening for indels, we have the learning resources, technical information, products, and technical support team to ensure the success of your CRISPR/Cas9 genome editing project at every step.

•

In vitro sgRNA production—Quickly generate templates for in vitro transcription of any sgRNA, without ligation, and produce high yields of sgRNAs

•

Testing sgRNAs—Determine the efficacy of individual sgRNAs in vitro before introducing them into cells

•

Delivering sgRNA & Cas9 into cells /in vivo—Cas9/sgRNA co-expression vectors allow simple insertion of your target sequence and express ultra-bright fluorescent markers

•

Confirming Cas9 expression—Our super-sensitive polyclonal antibody detects wild-type Cas9 and commonly used variants

•

Ensuring editing—Our robust mismatch detection method outperforms CEL1-based assays

•

Confirming genotype—Determine if a given clone has mutations on one allele (monoallelic), both alleles (biallelic), or is unchanged (wild type)

•

Identifying indels—Characterize CRISPR/Cas9-introduced mutations with a simple four-step protocol

Learn more at www.clontech.com/Guide-it or contact tech@takara-clontech.eu Takara Bio Europe

orders@takara-clontech.eu • tech@takara-clontech.eu • Europe: +33 1 3904 6880 • Austria: 0800 296 141 • Germany: 0800 182 5178 Switzerland: 0800 563 629 • United Kingdom: 0808 234 8063 For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Clontech®, the Clontech logo, Guide-it, and that’s GOOD science! are trademarks of Clontech Laboratories, Inc. Takara and the Takara logo are trademarks of TAKARA HOLDINGS, Kyoto, Japan.

All other marks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. © 2015 Clontech Laboratories, Inc.

www.clontech.com

ADVERTORIAL

Setting up a Bio Science business – Intellectual Property

This is the first in a series of short articles on aspects of starting a bio science business Although some very successful service businesses have been built without specific intellectual property and instead have relied on efficient delivery or the ability to source particular materials or skills most businesses in this space are built on a foundation of exclusive rights to a particular technology secured by patents and supporting intellectual property. Accordingly the first step in setting up a new business is usually to assess what intellectual property will be needed in the business and then to secure the necessary rights.

Firstly you need to review the competitive landscape to see if others are working in your field or own patents that could block your pursuing your idea. You may be able to do this initial search yourself but if you are anxious or if your own search turns up potentially conflicting patents you should engage a patent agent with specific expertise in your area of interest. If you are looking to spin out from a university or other business it makes sense to choose an agent who does not work for your current employer. Explain your proposed business to the agent and they will be able to review any potentially blocking patents and advise if there is a way through for you and also on the potential to patent your own ideas.

At this stage you will not want to spend a lot of money so the searching will be at a fairly high level but will generally be sufficient to give you an idea as to whether there is going to be sufficient freedom from the rights of others to take your ideas forward.

The next step is to secure rights to any third party patents that may be standing in your way or that you want to use in the business.

For this you need to engage a specialist lawyer – again be sure to find someone who understands the industry and make sure you brief them in detail as to the business you have in mind so that they can give you the most appropriate advice. You can either buy the rights outright – an assignment – or take a licence to the technology in whole or just in relation to the field in which you are working. Universities generally want to see their technologies exploited so are open to discussing licensing but will not usually assign existing technology to a new company for fear that the company will fail and the technology will not be exploited however it may be possible to include an option to buy the university out once the business is more established. Remember if your idea arose in the course of your employment it will usually belong to your employer and there

may be other restrictions in your contract of employment that limit your right to set up a business in competition to your employer.

Read your contract carefully and show it to your lawyer.

There is a tendency to focus on patents but other types of intellectual property may be available; in particular do not overlook the significance and potential weaknesses of copyrights for software based businesses or the potential to exploit databases to develop your business. While trademarks are generally not important at this early stage they can be worth considering later as goods and services approach the market.

By Patricia Barclay Bonaccord

Bonaccord – UK Life Science Law Firm of the Year www.bonaccord.eu

The CRISPR conundrum With CRISPR research accelerating faster than any other field in life science, how can your lab take advantage of the latest best practices for sgRNA and library design?

Starting today, if you wanted to get up to speed on all-things CRISPR, you would need to read 45 papers every week for a year, or 20 papers per week if you were already up to speed. That burden would double every year after.

While challenging, finding the time to read papers is important in helping your lab stay up to speed with the latest techniques in the fast-moving CRISPR field. However, every day researchers’ experience with the technique deepens and biologists are finding new methods to design more effective guides (gRNAs,) or employ Cas9 variants in imaginative ways.

In particular, the field is continually identifying new selection rules for identifying ideal gRNAs to perform specific kinds of experiments in different species, tissue types and cell lines – all of which can vary based on your experimental intent (knock-out, knock-in, deletion etc). The conundrum is clear: with the field moving faster than you can keep up with on your own, getting the best out of CRISPR often seems out of reach.

Figure 1. DESKGEN supports a variety of approaches including nickase pair mode, where non-viable paired gRNAs are excluded automatically

GUIDE SELECTION RULES AND THE EXPLOSION OF CRISPR SOFTWARE TOOLS

• Donor design – one-click donor design with PAM site scrambling and homology arm length variation

To solve this problem, academic labs have built software tools that capture their findings as algorithms that can be applied elsewhere. This trend has seen a multitude of software tools emerge, with each offering a different approach to one or two aspects of gRNA design. Unfortunately, these tools are not currently integrated for your use in a single place.

For instance, the Broad Institute sgRNA Designer ( Doench et al. 2014) calculates an “on-target activity score” for a gRNA based on its nucleotide composition, as certain nucleotides affect the cutting activity of the CRISPR system. The on-target score effectively determines if gRNAs will be effective at their intended cut site. On the other hand the MIT Tool’s off-target activity score (Hsu et al. 2013) calculates the likelihood and severity of off-target effects based on similar sequences occurring in the genome. It considers mismatch position and density between the gRNA sequence and the potential off-target sequence. Good guides must exhibit high enough activity to disrupt the gene, without having detrimental off-target hits elsewhere. While both selection rules are important, they are rarely considered tog.

Other useful features are also variably distributed across different tools. For instance, the Sanger Institute tool provides a genome browser but no gRNA selection table, whereas CAS Blaster is the opposite.

With each software tool being well suited to the specific use case for which it was designed, expertise are fragmented across different features and tools, making the ideal experimental design process (and steps to reproduce data) unnecessarily difficult.

DESKTOP GENETICS PROVIDES MULTIPLE SOLUTIONS TO THE CRISPR CONUNDRUM Its important to use the latest sgRNA design criteria, but reading all the papers and testing all the tools until you find the sgRNAs you want is not feasible.

The DESKGEN platform, by Desktop Genetics, is a computational toolkit that provides all of the functionality most important in ensuring successful and hassle-free genome editing. Our algorithms combine on-target and off-target scoring functions, assess relevant sequence characteristics, and make

• Automate vector design – easy oligo design to clone your guides into any vector • Integrated genome browser – supporting ENSEMBL data Thousands of scientists are already benefiting from the DESKGEN public platform. To find out more visit www.deskgen.com Figure 2. In a head to head comparison DESKGEN finds more off-target hits than the MIT CRISPR tool use of experimental data and machine-learning approaches to produce an optimal guide list, so that you don’t waste time on guides destined to fail.

We work with leaders from academia and industry and are constantly updating our CRISPR algorithms and features to let you can take advantage of stateof-the-art practices from leading genome engineering laboratories. Our platform can be used in two ways: 1) Directly in your lab on our public platform, available at deskgen.com (fig. 1) 2) By our in-house bioinformaticians to design custom libraries with enhanced performance (see next page)

1) THE DESKGEN PUBLIC PLATFORM Available for free for academics, and as an enterprise deployment for companies, our platform provides you with the ability to design, score and visualise gRNAs in a high definition genome browser (fig. 1) that supports dedicated modes for knocking out, knocking in and much more: • Cas9 variants – all popular Cas9 orthologues & PAM sites supported • Cpf1 nuclease - for AT-rich genomes and regions • Multiple genomes – 20+ including plant, bacteria & model organisms • Guide-filtering – auto-exclude guides based on GC & homopolymer content • Off-target scoring – finds more off-target hits than any other tool (fig. 2) • On-target scoring – determines predicted gRNA activity for all Cas9 variants • Variable gRNA length – pick standard or truncated guides

2) DTG LIBRARY SERVICE While our public platform provides you with everything you need to get started, you can always benefit from an expert’s touch - especially when there are so many variables to consider, or if you are only just getting started in CRISPR. Further, every cell line has a unique genome, and every target in that genome is unique - which is why we also offer library design services libraries using the exact sequence of your particular cell line, and not the reference genome.

To create our libraries, our bioinformaticians run an advanced version of the platform that makes use of additional scoring rules and expertise that we have forged whilst working with leaders in the field including Editas Medicine, enEvolv and Horizon Discovery. Our libraries allow you to: • Design less guides per gene, meaning more targets can be screened for less cost and time • Achieve high specificity, meaning more targeted experiments can be performed with confidence • Reduce confounding side effects, for instance cell death-from unanticipated off-target mutations Right, we provide data on a recent DTG library depletion assay study which demonstrates enhanced library performance for knock-out applications (figures 3-5).

Our bioinformatics group will work with you to design the best guides for your high-throughput genomic screens for loss of function, target discovery, target validation, phenotypic and other functional genomic screens. You can get these guides synthesised and clone them yourself, or we can take care of it for you through our partnership with Transcriptic - just send us an empty CRISPR vector and we’ll clone the guides we design directly into it at market-beating prices and turnaround times.

Figure 3. DTG knockout library design process. 3500 sgRNAs were designed for a sgRNA depletion assay targeting 100 essential genes in AL375 cells. NAG PAM sites serve as negative controls, all NGG sgRNAs are then filtered into groups for: high on-target activity using Doench function; low off-target activity using an adapted Hsu et al. scoring function and additional filtering based on sgRNA sequence traits, GC content, homopolymer content. sgRNAs scoring well in all criteria are pooled as the “DTG library”.

Figure 4. By combining sgRNA design criteria, we demonstrate improved depletion rates in DTG’s combined libraries, indicating superior KO performance to libraries designed using one set of criteria alone.

Figure 5. Further quantification of most- and least-active sgRNAs demonstrates that DTG library rules provide more active and less inactive sgRNAs than implementation of single design rules in isolation.

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

Cell therapy manufacturing industry takes big steps forward

Cell Therapy production across the world has made some significant advances in recent months, with investment in manufacture announced and exciting research suggesting new applications.

permission to The Cell Therapy Catapult to construct the £55 million large-scale manufacturing centre.

advance the industry into becoming a world leader in advanced therapy development and commercialisation.

The 7200m2 centre, which is expected to open in 2017, will be managed by the Cell Therapy Catapult and be used for manufacture of products for late phase clinical trials and for the commercial supply of advanced therapeutic medicinal products including cell and gene therapies.

“The large-scale GMP manufacturing centre will provide global scientific and medical communities with the assistance they need to turn research into products that have the potential to address many unmet medical needs.”

The facility is expected to create up to 150 jobs and its position on the Stevenage Bioscience Catalyst campus will provide additional inward investment from global companies.

The centre comes after the company’s 2013 survey of the UK’s manufacturing capability identified limitations in manufacturing and the supply chain as one of the barriers to the translation of research into commercially viable products.

The industry in the UK has taken a step forward with confirmation that a new plant is to be built in Hertfordshire. Stevenage Borough Council has granted planning

Keith Thompson, CEO of the Cell Therapy Catapult, said: “I am delighted that Stevenage Borough Council has granted permission to create a centre that will

The Cell Therapy Catapult was established in 2012 as an independent centre of excellence to advance the growth of the UK cell and gene therapy industry, by bridging

44

BIOSCIENCE JOURNAL WINTER 2015

FEATURE z

the gap between scientific research and fullscale commercialisation. It has more than 100 employees focusing on cell and gene therapy technologies.

Therapeutics has announced encouraging results for its T-cell receptor (TCR) therapeutic targeting the NY-ESO-1 cancer antigen in patients with multiple myeloma.

In another development, The Cell Therapy Catapult has awarded a manufacturing contract to Manchester-based company Cellular Therapeutics Limited (CTL) to help develop an engineered T cell treatment for blood cancers such as acute myeloid leukaemia and myelodysplastic syndrome.

Multiple myeloma is a cancer formed by malignant plasma cells. Normal plasma cells are found in the bone marrow and are an important part of the immune system, which is made up of several types of cells that work together to fight infections and other diseases. Multiple myeloma is characterised by low blood counts, bone and calcium problems, infections and kidney problems.

The contract will support Phase I and II trials already under way, which involve the genetic modification of patients T cells so that they recognise and destroy WT1expressing cancer cells when infused back into the body.

CTL’s work will complements research being carried out in collaboration with Imperial College London, University College London and Great Ormond Street Hospital, led by Prof Hans Stauss and Dr Emma Morris, and originally funded by the charity Leukaemia & Lymphoma Research.

According to The Cell Therapy Catapult, demand for immune cell therapy manufacturing is rapidly expanding globally and Keith Thompson said: “This contract will greatly enhance our WT1 clinical programme and accelerate this research intended to benefit patients with these life-threatening disorders.”

AMERICAN RESEARCH PRODUCES ENCOURAGING RESULTS In America, clinical trials have offered another cause for optimism for the field.

US biopharmaceutical firm Adaptimmune

Encouraging clinical responses were observed in 16 patients (80 per cent) in the study: Of the 20 patients, 14 patients (70 per cent) had a near complete response or complete response, and another two had a very good partial response three months after treatment.

Dr Rafael Amado, Adaptimmune’s Chief Medical Officer, said: “We believe these are significant data for Adaptimmune and for the cancer gene therapy field.

“The trial showed that autologous transduced cells can be safely administered to patients with advanced myeloma in the context of stem cell transplantation, and that the transduced cells persist for a prolonged period of time. There was also encouraging evidence of anti-tumour effect which supports further investigation of cell and gene therapy in myeloma.”

Aaron P. Rapoport, MD, the Gary Jobson Professor in Medical Oncology at the University of Maryland School of Medicine and the Director of the Blood and Marrow Transplant Program at the University

45

of Maryland Marlene and Stewart Greenebaum Cancer Center, said: “This study establishes a strong foundation for further research in cellular immunotherapy of myeloma.

“We hope to investigate additional combination approaches to boost the durability and function of the engineered T-cells to achieve even longer and deeper clinical responses.”

JAPAN OFFERS GREAT POTENTIAL FOR CELL THERAPY MANUFACTURE

A further boost to the field came when Lonza, the world’s leading developer and manufacturer of cells for regenerative medicine therapeutics, and Nikon Corporation announced an exclusive collaboration in the field of cell and gene therapy manufacturing in Japan.

Japan has become an attractive location for the future of regenerative medicine since the induction of the Revised Pharmaceutical Affairs Act, which became effective in November 2014. The act states that conditional product approval may be granted in Japan if clinical safety and an indication of efficacy of a regenerative medicine product are demonstrated.

Under the agreement, Nikon will acquire the technical know-how to differentiate and manufacture cells, including somatic stem cells and Andreas Weiler, Head of Emerging Technologies, Lonza Pharma&Biotech. said: “This collaboration will contribute greatly to the growth of the global cell and gene therapy market.”

BIOSCIENCE JOURNAL WINTER 2015

ADVERTORIAL

Cell therapy manufacturing – making the future now There is no mistaking the sure but steady revolution happening in cell therapy. With the long-held promise now rewarded with greater funding, investment and belief, the change in gear has been dizzying.

However, this is merely the end of the beginning and the hurdles we must overcome in disseminating clinical manufacturing are surely the next beach-head. Not just for now but how cell products can be produced as the scale of cell therapy matures through its adolescence.

Miltenyi Biotec has been involved in cell therapy development for nearly twenty years.

It has always been clear that cell manufacture will require a closed-system, independence from higher-grade operating backgrounds, automation, standardisation and single-device processing. This will be increasingly obvious and of vital concern as cell therapy progresses to Phase III trials and on to commercialisation.

Miltenyi Biotec’s vision has thus evolved to provide closed-system cell manufacture in one device with all required GMP materials. With larger-scale GMP cell manufacturing becoming real, the advantages of having one experienced partner providing all requirements in a facilitating manner are becoming clear, exclusive licencing being another growing concern in this changing market.

The CliniMACS Prodigy® is one of the first truly closed, automated end-to-end GMP cell processing devices and its potential is now beginning to be realised.

The unmistakable poster boys of cell therapy right now are undoubtedly CAR T cells, with such promise in the clinic and burgeoning interest from Big Pharma and private

IPC/QC

Donor/ patient Cryopreservation

Blood leukapheresis

Sample preparation

T cell selection

IPC/QC

IPC/QC

Activation

Cryopreservation

Expansion

Final formulation

IPC/QC

Administration to patient

IPC/QC

Figure 2: Workflow for production of gene-engineered T cells (adapted from Kaiser et al. (2015) Cancer Gene Therapy 22: 72–78.) investment. However, production of these cells is long and complex with many different steps, including the utilisation of genetically modified viral vectors. To encompass this process into a closed automated device is perhaps the key to any organisation wishing to lead the field in this arena. It is thus exciting that the CliniMACS Prodigy T Cell Transduction Process is about to be released – a fully integral manufacturing solution in a closed-system.

A process comprising preparation, isolation, activation, viral transduction, expansion and final formulation, closed and automated, which is flexible enough to allow subtleties in differing protocols and optimisations but rigid enough to maintain GMP fidelity. An end-to-end solution that can be monitored and controlled.

While the field becomes aware of the requirement of such processing, we believe the CliniMACS Prodigy is leading the way. As cell manufacturing progresses, the main difference we shall see will be in scale. Many processing solutions for allogeneic cells (e.g. MSCs) have necessarily focused on increasing cell numbers and establishing cell banks. However, personalised autologous applications, such as CAR T cells, rather need an increasing number of individual products. This is where such a system comes into its own, particularly where viral vectors are concerned.

Figure 1: The CliniMACS Prodigy (Image copyright © 2015 Miltenyi Biotec GmbH. All rights reserved.)

Transduction

Enrichment of modified T cells

A fully closed device providing an end-to-end process can give independence from facility capacity. Currently, only one product is usually produced in one cleanroom at a time, but a closed-system means that multiple devices can operate in the same room simultaneously.

46

Capacity then becomes a mere factor of how many automated devices are running, with all the associated benefits to operational costs and management.

The benefits of device-based manufacturing are clear over any production line using multiple facilities and varying devices.

A centralised production model is then easier to establish, but because the devices are automated and so easily transferable it also applies to a localised or point-of-care model.

A smaller number of devices at local units enables manufacture of personalised cell products in the same standardised way.

The CliniMACS Prodigy is a GMP cell manufacturing platform made truly integral with our GMP raw materials, which include now, thanks to our new colleagues from Lentigen Technology Inc., lentiviral vectors.

GMP cell processing also requires thorough QC and analysis of cell products. For a complete solution we developed the MACSQuant® Flow Cytometers and flow reagents, bracketing the closed-system manufacturing with highperformance analysis.

It is exciting to see cell therapy finally begin to blossom and to see what comes next. We believe that in the CliniMACS Prodigy we have a complete, compact solution, easily transferable and instantly scalable, for the needs of ATMP production just now being perceived.

By Jason Jones

Manager, Cell Therapy, Miltenyi Biotec Ltd.

Automate your manufacturing

Your trusted partner in cell and gene therapy • Fully integrated workflows for ex vivo cell processing • Established protocols for gene modification of T cells and stem cells • Design, development, and manufacturing of customized lentiviral vectors • Commercial-scale supply of ancillary materials for manufacturing

miltenyibiotec.com/prodigy Miltenyi Biotec Ltd. | Almac House, Church Lane | Bisley, Surrey GU24 9DR | UK Phone +44 1483 799 800 | Fax +44 1483 799 811 | macs@miltenyibiotec.co.uk The CliniMACS® System components, including Reagents, Tubing Sets, Instruments, and PBS/EDTA Buffer, are manufactured and controlled under an ISO 13485–certified quality system. In the EU, the CliniMACS System components are available as CE-marked medical devices. In the US, the CliniMACS CD34 Reagent System, including the CliniMACS Plus Instrument, CliniMACS CD34 Reagent, CliniMACS Tubing Sets TS and LS, and the CliniMACS PBS/EDTA Buffer, is FDA approved; all other products of the CliniMACS Product Line are available for use only under an approved Investigational New Drug (IND) application or Investigational Device Exemption (IDE). CliniMACS MicroBeads are for research use only and not for human therapeutic or diagnostic use. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. CliniMACS, CliniMACS Prodigy, and MACS are trademarks or registered trademarks of Miltenyi Biotec GmbH. Copyright © 2015 Miltenyi Biotec GmbH. All rights reserved.

BIOSCIENCE JOURNAL WINTER 2015

ADVERTORIAL

Cell therapy manufacturing Regenerative medicine and cellular therapies have the potential to impact many different areas of unmet medical need. There are many human diseases which have either inadequate or no treatment options. One of the main focuses of cellular therapy is the use of stem cells to treat diseases such as osteoarthritis or Critical Limb Ischemia – these products are defined as Advanced Therapeutic Medicinal Products (ATMPs).

Cellular therapy is not a technology of the future, it is having an impact now, with thousands of on-going clinical trials using stem cells as the preferred treatment option.

For cellular therapies two distinct categories exist- autologous and allogeneic. Autologous is using the sick patients owns cells and modifying or expanding them and then injecting these cells back into the patient i.e. one patient treatment. Allogeneic is whereby healthy donor cells are used to manufacture the treatment product and injected into the sick patient and/or patients i.e a multirecipient dose which allows for a more conventional ‘off the shelf product’.

A cellular Therapy clinical trial is a complex multidisciplinary endeavour, requiring

coordination between scientists, clinicians, and the GMP manufacturing site. There are many stages in a cellular therapy clinical trial which are all inextricably linked; donor recruitment, screening & tissue/ cellular procurement, manufacturing at the GMP manufacturing site, product characterisation & batch release, and distribution back to the clinical site for administration to the clinical trial subject. Each element must be well-defined and rigorously controlled in order to ensure that the cellular product is consistently safe and efficacious, prior to injecting into the patient.

One of the biggest challenges is the provision of an authorised manufacturing facility for the supply of these cellular products. Authorisation from the national regulatory body must be obtained in order to manufacture such products. The manufacturing of cellular products is unique in that it is not a conventional pharmaceutical product but it is a biological product which is manufactured from a living tissue source (starting material). Although it is nonconventional in a pharmaceutical context, the manufacturing of these still require the same level of rigor and overall GMP regulatory requirements. As the starting material is of a human source, this can lead to variability in the manufacturing of the product and batch to batch variation, although the same release criteria must be applied to each batch.

The sustainability of these manufacturing facilities is another key challenge, where many of these were set up as translational facilities within a University setting. While the traditional pharmaceutical industry is driven primarily by profit and fulfilling shareholder

48

BIOSCIENCE JOURNAL WINTER 2015

needs, the ATMP facilities are largely funded through governmental funding at local and international levels and this may include funding from the University or Institution, where manufacturing facility is located. These facilities are largely focused on patient benefit and act as translational facilities from the bench to the bedside. The focus is not profit driven for the majority of such ATMP facilities and so the challenge of maintaining and running these pharmaceutical facilities is significant.

The same level of rigor and compliance to GMP as in a conventional pharmaceutical facility is required in these smaller non-profit operations.

Another key challenge for manufacturing these products, is the availability of competent stem cell experts, who play a critical role at key decisions points in the process. The manufacturing process requires these highly qualified and trained personnel to produce these cells, and these operators are also

ADVERTORIAL

required to perform routine/typical cleanroom tasks such as cleaning and environmental monitoring. It follows that there is the added challenge of retaining these people who perform significant scientific processes but yet have to perform the routine day-to-day cleanroom activities.

One such ATMP facility is the Centre for Cell Manufacturing Ireland (CCMI) at the National University of Ireland Galway (NUIG), which is a custom-built licensed facility designed to manufacture Advanced Therapeutic Medicinal Products such as stem cells for use in human clinical trials. CCMI has facilitated the pathway from the bench to bedside with a focus on GMP manufacture, product characterisation, stability of the IMP and distribution to the trial site, for patient treatment. CCMI received Irish Regulatory Approval in August 2013 to manufacture human Mesenchmyal Stem Cells (hMSCs) for clinical use. CCMI is the first and

49

only approved facility on the Island of Ireland that can manufacture (expand) these hMSCs for clinical use.

CCMI is currently manufacturing hMSCs for a clinical trial for Critical Limb Ischemia, for which the regulatory body in Ireland, the Health Products and Regulatory Authority (HPRA) gave approval for in 2014. This trial will treat at least nine patients, which will require the manufacture of nine human mesenchymal cell batches by CCMI over the period 2015 to early 2017. There are three further clinical trials in the pipeline for CCMI in 2016/2017; 1. ADIPOA-2: Autologous Adipose Derived Stem Cells (ASCs) for the treatment of osteoarthritis 2. Nephstrom: Allogeneic hMSCs for the treatment of diabetic kidney disease 3. Visicort: hMSC in Corneal transplant. www.nuigalway.ie/stem-cells

Ireland’s first Centre for Stem Cell Manufacture

The Centre for Cell Manufacturing Ireland (CCMI) at the National University of Ireland Galway (NUIG) is authorised by the Health Products and Regulatory Authority (HPRA) – (Irish Regulatory Body) for the manufacture of Human Mesenchymal Stem Cells for clinical trials.

CCMI is a versatile cell manufacturing facility with standout clean room features including two independent parallel production suites. Each of the two suites is capable of clinical grade manufacturing of cellular therapy products and small molecules for therapeutic applications. The facility is served by dedicated utilities including CO2, on site Liquid Nitrogen generation and HEPA filtered air. The facility is custom-designed with validated HEPA air system to isolate manufacturing, intermediate and gowning/de-gowning rooms from each other. Local isolation areas are of EU GMP Grade A air quality,

For further information please contact: Andrew Finnerty, General Manager T: +353 91 494159 F: +353 91 495547 E: andrew.finnerty@nuigalway.ie

in both suites. Each suite is equipped with fully-validated equipment, necessary for the production of the clinical products.

The environment within the facility is managed using a facility monitoring system.

Each suite is controlled by separate air handling units and so this can facilitate the production of several different product types at the same time e.g. allows the production of conventional cellular products or cellular products whereby containment is required. The facility is staffed by dedicated highly skilled personnel and has implemented

www.nuigalway.ie/stem-cells

a Quality Management System (QMS) to ensure full compliance with EU legislation for manufacture of cell based IMP. CCMI are interested in discussing opportunities with clients who are seeking to procure cellular therapy products, for example CAR therapy products and NK Cells. CCMI can tailor the cell manufacturing process to the customer’s needs and will work with these clients on transferring processes and seeking required regulatory approvals. CCMI will manufacture ATMP according to a specified client protocol and related SOPs.

At the UK’s largest single-site university, we possess a fantastic breadth and depth of knowledge. We break down barriers to undertake multidisciplinary research that can help you achieve your objectives and face future challenges.

Find out more about working with us:

 0161 275 2175  collaborate@manchester.ac.uk  www.manchester.ac.uk/collaborate

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

Better understanding of

immunology

offers hope for the future Recent years have seen dramatic strides being taken in the science of immunology – the capacity to direct the body’s natural defences against specific illnesses and conditions.

So dramatic have the strides been that scientists’ thoughts have increasingly been turning from the short-term suppression of symptoms to treatments that act over a much longer term or even possibly to cures.

Central to the developments have been a deeper understanding of the way the immune system works with clinicians, researchers and drug companies increasingly able to bring relief to sufferers of conditions ranging from rheumatoid arthritis to cancer.

Immunology is a branch of biomedical science that covers the study of all aspects of the immune system and today’s practitoners are building on thousands of years of scientific development.

Indeed, the idea of immunity dates back at least to Greece in the 5th century BC when Thucydides observed people who recovered from the plague in Athens and became immune to re-infection.

The earliest recognised attempt to use that knowledge took place in 10th Century China, where healthy people were exposed to lesions caused by smallpox to develop immunity.

Succeeding centuries saw more advances including in Britain when, in the 18th Century, the surgeon Charles Maitland exposed six condemned prisoners to smallpox. When they survived, the practice spread rapidly throughout England in the 1740s then to the American colonies.

Other key figures were Edward Jenner, for development of cowpox as a safe vaccine for smallpox, Robert Koch, a 19th Century country physician who developed

techniques necessary for the cultivation of bacteria, and Louis Pasteur, another pioneer in the field of vaccines.

The past century, and in particular the past two decades, has seen scientists use all that accrued knowledge to they gain a better understanding of the primary lymphoid organs of the immune system the thymus and bone marrow, and secondary lymphatic tissues such as spleen, tonsils, lymph vessels, lymph nodes, adenoids, and skin and liver.

Many components of the immune system are cellular in nature and not associated with any specific organ but rather embedded or circulating in various tissues located throughout the body and research has cast more light on how that process works.

Dr Alan Worsley, Senior Science Communications Officer at Cancer Research UK, said: “We have become more fully aware of the potential of using the immune system over the past hundred years and the rate of development has really accelerated over the past fifteen to twenty years.

“Previously, we saw the immune system as an on-off switch but now we see it more as a fusebox with start, stop, slow, accelerate switches and we are beginning to understand the complexities of the immune system, the checks and balances. We’ve made a lot of headway and, although we are starting to understand much more, there is still a great deal left to do.

“The immune system is designed to attack anything in the body that should not be there, such as the flu virus. But it is much more challenging when the immune system has to deal with a threat that it recognises as a normal part of the body.

52

“Take the rogue cells that cause cancer as an example.

We have to find ways to help the immune system recognise them as something that should be eradicated or inhibited even though they are a natural part of the body and drugs are coming on the market that can do that.

“In terms of where we are with progress, I would liken it to the history of electronic devices where we started with the lightbulb and have reached the smartphone. In the field of immunology I would say that we have reached the colour television.

“However, the process is accelerating and I see it doing so at a rapid rate in the years to come.”

A similar picture is painted by Professor Michael Ehrenstein, a British Society for Immunology spokesperson and Professor of Rheumatology at University College London, who said: “We are certainly seeing rapid development in the science, driven in part by the successes we are having as we learn to target treatments.

“Our understanding of the immune system is developing all the time as we see which treatments work. There is improved communication between researchers and clinicians so that good feedback is provided.

It is very much a two-way street.

“What is also helping is that the drugs companies are seeing the potential and investing in bringing forward new treatments, which means that clinicians have more options.”

BIOSCIENCE JOURNAL AUTUMN 2015

FEATURE z

We have become more fully aware of the potential of using the immune system over the past hundred years and the rate of development has really accelerated over the past fifteen to twenty years.

Dr Alan Worsley

Senior Science Communications Officer at Cancer Research UK

CONTINUED ON PAGE 54

53

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

CONTINUED FROM PAGE 53

Tackling

rheumatoid arthritis Professor Ehrenstein’s specialism is rheumatoid arthritis and he said: “Because we are becoming much better at targeting our treatments, we are much better at suppressing symptoms. We do not have a cure for rheumatoid arthritis yet but it’s something that people are working towards.”

Among work being done is that by a team at the La Jolla Institute for Allergy and Immunology in California in the United States, who recently revealed that they had developed a potential new treatment.

Working in collaboration with colleagues at the University of California, San Diego, they came up with a drug that focuses on the cells that are directly responsible for the cartilage damage in affected joints.

Rheumatoid arthritis, an autoimmune

disease that leads to stiff, deformed joints and often crippling pain, affects millions of people across the planet as the immune system attacks the body’s own tissue.

The inflammatory processes activate synoviocytes (FLS), cells that line the inside of joints. Once mobilised, the FLS invade the surrounding cartilage and secrete enzymes that break down the firm, rubbery tissue that cushions the bone. In addition, they trigger bone destruction.

54

Much of the current treatment focuses on intercepting the immune system’s misdirected attack on the lining of affected joints to alleviate the symptoms, reduce inflammation and slow the progression of the disease.

For the study’s lead author Nunzio Bottini, M.D. Ph.D., associate professor at La Jolla Institute and associate professor of Medicine at the University of California, San Diego, better understanding immunology is the key to advancment.

He said: “Unfortunately, for around 40 per cent of patients, immune-targeted therapies are not sufficient to bring them into full remission. If we could add a drug that acts on a different target without increasing immune suppression it could be very valuable.

“Even if your inflammation is completely under control with the help of current therapies - and they are excellent - the damage to the skeletal structure is not necessarily arrested in the long term because synoviocytes continue to cause damage and, although synoviocytes are considered the main effectors of cartilage damage in rheumatoid arthritis, there’s no therapy directed against them.”

That was until post-doctoral researcher and first author, Karen M. Doody, Ph.D., while screening samples from rheumatoid arthritis patients, discovered that an enzyme known as RPTPσ, short for receptor protein tyrosine phosphatase sigma, is highly expressed on the surface of FLS. Normally, RPTPσ is kept inactive but, when activated using a biological decoy, it weakens the ability of arthritic synoviocytes to aggressively invade the joint’s cartilage.

Co-author Gary S. Firestein, M.D., dean and associate vice chancellor of Translational Medicine and director of the Clinical and Translational Research Institute at UCSD, said: “The unique aspect of this approach is the ability to improve symptoms and decrease joint damage while potentially avoiding any negative effects on normal immune responses and susceptibility to infections.”

BIOSCIENCE JOURNAL WINTER 2015

FEATURE z

Everyday tablet could lead to relief for cancer sufferers Another example of immunology at work is new research that suggests that giving cancer patients aspirin at the same time as immunotherapy could dramatically boost the effectiveness of the treatment.

Francis Crick Institute researchers, funded by Cancer Research UK, have shown that skin, breast and bowel cancer cells often produce large amounts of prostaglandin E2 (PGE2).

This molecule dampens down the immune system’s normal response to attack faulty cells, which helps cancer to hide. It is a trick that allows the tumour to thrive and may explain why some immunotherapy treatments have not been as effective as hoped.

Study author Professor Caetano Reis e Sousa, senior group leader at the Francis Crick Institute, said: “We’ve added to the growing evidence that some cancers produce PGE2 as a way of escaping the immune system. If you can take away cancer cells’ ability to make PGE2 you effectively lift this protective barrier and unleash the full power of the immune system.

“Giving patients COX inhibitors like aspirin at the same time as immunotherapy could potentially make a huge difference to the benefit they get from treatment. It’s still early work but this could help make cancer immunotherapy even more effective, delivering life-changing results for patients.”

Professor Peter Johnson, Cancer Research UK’s chief clinician, said: “PGE2 acts on many different cells in our body, and this study suggests that one of these actions is to tell our immune system to ignore cancer cells. Once you stop the cancer cells from producing it, the immune system switches back to ‘kill mode’ and attacks the tumour.

Aspirin is part of a group of molecules called COX inhibitors, which stop the production of PGE2 and help reawaken the immune system. Combining immunotherapy with aspirin or other COX inhibitors substantially slowed bowel and melanoma skin cancer growth in mice, compared to immunotherapy alone.

“This research was carried out in mice so there is still some way to go before we will see patients being given COX inhibitors as part of their treatment.

But it’s an exciting finding that could offer a simple way to dramatically improve the response to treatment in a range of cancers.”

Flu outbreaks teach valuable lessons A study of influenza is also helping scientists to learn more about the immune system.

A four-year study of 1,414 unvaccinated people across England found that 43 per cent had immune cells that protected them from symptoms of both seasonal and pandemic influenza.

The work led by researchers from UCL, Oxford University and Public Health England, funded by the Medical Research Council and Wellcome Trust, showed that certain T cells, immune cells that fight infection, can help to control influenza infections by targeting a protein common to all strains of influenza A. Influenza A is the most common type of influenza and is the only type that can cause pandemics The new finding offers the possibility of

a universal vaccine to reliably reduce the severity and spread of all types of influenza A. Professor Andrew Hayward, of UCL Farr Institute of Health Informatics Research, said: “Current flu vaccines help us make antibodies that target the proteins on the outside of a flu virus. These evolve gradually from year to year and dramatically in the event of a pandemic making it hard for the public health community and vaccine manufacturers to keep up,” “This was

55

illustrated last year, when the seasonal flu vaccine was much less effective than normal. It’s also why we don’t have vaccine available at the start of a flu pandemic when it would be most useful.

“Although a vaccine to boost flu-killing T cell responses would not prevent individuals from becoming infected in the first place, it would help to stop those who were infected getting ill and spreading the virus through coughs and sneezes.”

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

Inhaler technology developing all the time

For many years, respiratory diseases have been treated with drugs delivered via inhalers.

Sufferers from asthma and chronic obstructive pulmonary disease (COPD) are extremely familiar with the technique but research is now advanced on using the same pathway as a route for delivering drugs for the treatment of a wider range of conditions.

Cystic fibrosis, neurological conditions, bacterial infections and even cancers have been mooted as possible targets, as well as pain management and vaccines.

Additionally, existing drugs delivered in this way could have different effects from those they demonstrate when administered by other routes. For example, the anti-depressant amitriptyline, by blocking ceramide levels, has the potential to treat inflammation and infections in the respiratory tract.

Using the lungs as a delivery pathway has both advantages and challenges. On the negative side, the lungs are powerfully resistant to allowing molecules to cross into the bloodstream as a necessary immune response but, conversely, they provide a large area for absorption with easy access to the blood supply.

If drugs can be made to enter the body via this route, it can reduce the size of the dose necessary. Lower doses have the benefit of reducing the incidence of side effects, a positive result for both patients and drug developers.

Administration via an inhaler rather than orally can also mean drugs act faster on the system, making it an attractive proposition where a rapid effect is desired, for example in pain relief or easing a craving for cigarettes.

In cystic fibrosis and bronchiectasis, resistance to antibiotics is becoming a problem for treatment of infections but a €50 million Europe-wide research project is being led by Queen’s University, Belfast, to produce inhaled antibiotics which are more effective.

The university said that the iABC (inhaled Antibiotics in Bronchiectasis and Cystic Fibrosis) consortium will develop new inhaled antibiotics to manage chronic lung infection, the main cause of disease and death in patients with cystic fibrosis and bronchiectasis.

The new antibiotics are to be trialled over a five year period and are expected to improve patients’ quality of life by reducing lung infections and flare ups, improving lung function and overcoming antibacterial resistance which frequently occurs in patients with these conditions.

Professor Stuart Elborn, Dean of the School of Medicine, Dentistry and Biomedical Sciences at Queen’s University, and lead researcher on the project, said: “There are limited antibiotics available to treat lung infection in cystic fibrosis and bronchiectasis, and the bacteria causing them are becoming increasingly resistant to current antibiotics.

“This work has the potential to deliver inhaled antibiotics that will improve the quality of life and survival of cystic fibrosis and bronchiectasis patients.”

In addition, work is under way to treat diabetes with inhalers, seen by some as particularly useful for sufferers who find injecting themselves stressful. Inhaled insulin has gone on sale in the US this year for patients with both type 1 and type 2 diabetes.

Because inhalation drug development is mostly not about new compounds rather new delivery options it is known as repositioning and presents many engineering problems which must be overcome to devise workable inhalers which deliver the correct dose to the correct portion of the respiratory system in a reliable way which is acceptable to patients and easy for them to use.

Engineers are working on everything from the type of plastic used to the speed of delivery.

Four types of system are widely used; metered dose inhalers (MDI), dry powder inhalers (DPI), liquid droplet inhalers (LDI) and nebulisers.

This work has the potential to deliver inhaled antibiotics that will improve the quality of life and survival of cystic fibrosis and bronchiectasis patients.

MDIs are the market leaders with almost half of sales but DPIs are growing in popularity, partly because they do not rely on a propellant to deliver the dose.

Conferences and symposia are being held around the world every few months to keep people abreast of this exciting and rapidly developing field.

Patients with all kinds of conditions will increasingly be growing used to inhaling their medicine where in the past pills or injections were the norm.

Professor Stuart Elborn

Dean of the School of Medicine, Dentistry and Biomedical Sciences at Queen’s University

56

BIOSCIENCE JOURNAL WINTER 2015

FEATURE z

57

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

Bionic technology

that is pushing the boundaries of what is possible

The loss of a limb through illness or accident can be a truly devastating blow and, until recent years, prosthetics did not really offer much in the way of compensation for sufferers. False limbs tended to be clunky and clumsy, providing only basic functions and rudimentary in their construction.

That is now changing as the science of bionics advances at a rapid rate. Indeed, one of the biggest challenge when considering the developments is separating fact from science fiction. Increasingly, the two tend to blend into each other.

Despite advances which have produced more effective prosthetics, there remains much to be achieved, with the biggest challenge the creation of prosthetics that are fully controlled by the brain.

Nevertheless, for a scientific field that is only sixty years old, bionics has made dramatic progress.

Credit for the creation of the term “bionics” seems to lie with Jack. E. Steele, a worker at the Aeronautics Division House at the WrightPatterson Air Force Base in Dayton, Ohio, in the United States in 1958.

was the basis for The Six Million Dollar Man, the TV series which, while highly imaginative in its representation of a man who was rebuilt after a terrible accident, was instrumental in popularising the concept.

In the years that followed, the gap between fact and fiction has narrowed as scientists develop devices that edge closer to mimicking the body’s natural functions.

Steele was a medical doctor and Air Force Colonel and when he coined the term he was not referring to the concept of bionics as it has been popularised but to the study of biological systems and organisms to find solutions to problems in engineering.

His work, and the new word, attracted the attention of science fiction writer Martin Caidin, who in 1972 wrote a book called Cyborg, which referenced Steele. This book, in turn,

58

BIOSCIENCE JOURNAL WINTER 2015

FEATURE z

Exciting times as developers reveal breakthroughs

Nicky Ashwell

Everywhere you look, there is cause for optimism with a number of companies developing devices designed to make life easier both for those who have lost limbs and those who have lost senses such as sight.

Some of the recent breakthroughs have come from Leeds-based prosthetics company Steeper, which recently launched the smallest version to date of its prosthetic hand.

Developed using Formula 1 racing car technology, the bebionic small hand is built around a skeletal structure with miniaturised components designed to provide the most true to life movements.

The company says that the bebionic small hand marks a turning point in the world of prosthetics as it perfectly mimics the functions of a real hand through 14 different grips and its size makes it ideal for women, teenagers and small-framed men.

Nicky Ashwell, of London, became the first UK user. The 29-year-old, who is a Product Manager at an online fashion forecasting and trend service, was born without a right hand.

Before being fitted with the bebionic small hand, she used a cosmetic hand without movement; as a result, Nicky learned to carry out tasks with one hand.

The bebionic small hand has been a major improvement to her life, enabling her to do things previously impossible with one hand such as riding a bike, gripping weights with both hands, using cutlery and opening her purse.

She said: “When I first tried the bebionic small hand it was an exciting and strange feeling; it immediately opened up so many more possibilities for me.

The technology comprises a unique system which tracks and senses each finger through its every move – mimicking the functions of a real hand.

Development follows seven years of research and manufacturing, including the use of Formula 1 techniques and military technology along with advanced materials including aerograde aluminium and rare Earth magnets.

“I realised that I had been making life challenging for myself when I didn’t need to.

The movements now come easily and look natural. I keep finding myself being surprised by the little things, like being able to carry my purse while holding my boyfriend’s hand.

I’ve also been able to do things never before possible like riding a bike and lifting weights.”

Ted Varley, Technical Director at Steeper, said: “There’s a trend of technology getting more intricate and Steeper has embraced this. An accurate skeletal structure was firstly developed, with the complex technology then specifically developed to fit within this in order to maintain anatomical accuracy. In other myoelectric hands, the technology is developed first, at the expense of the lifelikeness.

The technology behind the bebionic small hand uses sensors triggered by the user’s muscle movements that then connect to individual motors in each finger and powerful microprocessors.

“The idea is to make prosthetics simpler to use.

Previously, the first two fingers in a prosthetic hand would be linked to the thumb, making for a pinch-like grip. What we are developing is prosthetics where each finger moves on its CONTINUED ON PAGE 60

59

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

CONTINUED FROM PAGE 59

own. The brain is a remarkable thing and will fill in the gaps by responding to the signals from the electrodes on the arm. That means that the user can do things like grip a wine glass or a piece of fruit without crushing it.”

Another UK company making great advances is Touch Bionics, a spin-out from the UK’s National Health Service, which has developed its own electrically-powered prosthetic hand with five independently powered fingers.

The company recently revealed its latest version, the i-limb quantum, which can change grips with a simple gesture and CEO Ian Stevens said: “The new hand combines unsurpassed functionality with design style.

It is smarter, faster, stronger and smaller than any of its predecessors.”

Rebekah Marine, an i-limb wearer, said: “I particularly appreciate the ability to almost effortlessly choose different grips using subtle but distinct gestures. The new extra small size will appeal in particular to female users and children.”

those with sight loss which is controlled by the eyes, and three Austrian men have become the first to undergo ‘bionic reconstruction’, enabling them to use a robotic prosthetic hand.

All three were victims of car or climbing accidents and suffered brachial plexus injuries, where damage occurs to the network of nerves that control movement and sensation in the upper limbs.

The latest development in the technique came from Professor Oskar Aszmann, Director of the Christian Doppler Laboratory for Restoration of Extremity Function at the Medical University of Vienna, and engineers from the Department of Neurorehabilitation Engineering of the University Medical Center Goettingen first requires amputation of the limb. This is then replaced with a robotic prosthesis which uses sensors that respond to electrical impulses in the muscle. Three months after amputation, all three men had much better movement in their hands.

What we are developing is prosthetics where each finger moves on its own.

The brain is a remarkable thing and will fill in the gaps by responding to the signals from the electrodes on the arm.

Ted Varley

Technical Director at Steeper

In the United States, scientists in San Jose, California, have developed a visual aid to help

Next fifteen years could see the biggest breakthrough of all Many of those involved in the bionics industry say that, impressive as the current breakthroughs are, much remains to be achieved and some of that will rely on the development of new technologies and new materials in the next few years.

Dr Kianoush Nazarpour, the man leading another pioneering bionics project, the biggest hurdle to success still remains the replication of the body’s natural functions.

Ted Varley, of Steeper, said: “It’s all about faster, lighter and more flexibility. Technology is developing all the time, including the creation of smaller motors which means that we can use two in the same prosthetic, increasing the power to do things.

“We are also looking at prosthetics hands where the wrist can rotate. The technology does exist to do that but it is not available to everybody. Everything we are doing is about making prosthetics as lifelike as possible.”

Lifelike is the key word and, according to

Funded by the Engineering and Physical Sciences Research Council, his team from the universities of Newcastle, Leeds, Essex, Keele, Southampton and Imperial College London, aim to develop electronic devices that connect to the forearm’s neural networks. That would allow two-way communications with the brain, giving amputees a limb that more closely mirrors the real thing.

Dr Nazarpour, who is part of Newcastle University’s Biomedical Engineering team, and who used to work for Touch Bionics, said: “Bionics is making rapid advances and the devices being brought forward are very impressive but the basic problem remains that we are a long way off a bionic hand that can communicate directly with the brain.

“The UK leads the way in the design of prosthetic limbs but the inability to

60

develop technology that allows the hand to communicate with the brain remains the main limiting factor.

“If we can design a system that allows this two-way communication it would help people to naturally reach out and pick up a glass, for example, whilst maintaining eye contact in a conversation, or pick up an apple without bruising it.

“At the moment, intelligent prosthetic hands can respond to sudden mechanical stimuli, eg they can re-grip. However, what we do not

BIOSCIENCE JOURNAL WINTER 2015

have are hands that relay this feeling to the brain so that the brain instinctively knows that the hand is doing.

“With our own hands we know that they are there without having to look at them or making a conscious decision every time we want to do something.

“That is because the hand is connected directly to the brain through nerves. What we are looking to do now is develop and use a sensor placed in the nerves in the remaining part of the arm to enable the prosthesis to talk and to listen to the brain so that it functions more naturally.”

What the team has already done is produce an early version of something reminiscent, they say, of Luke Skywalker’s artificial hand in Star Wars, based on the idea that electrodes in the bionic limb can wrap around the nerve endings in the arm. This would mean that for the first time the hand could communicate directly with the brain.

Dr Nazarpour said: “What we are seeking to do is fill in the gap between the prosthetic hand and the brain. To work, it requires sensors small enough to attach to the tip of the fingers to sense the world. We also need sub-milimeter electrodes to go into the foream nerves without triggering body’s immune system reaction. We know that pacemakers and cochlear implants work so the concept is sound.

FEATURE z

“We do not want something that will be rejected after a period of time by the body. The last thing we want is patients having to go through a series of operations to keep having it replaced.

“The sensors could also be used to help damaged livers regenerate or for someone with obesity by telling the stomach when it has had enough, eradicating the need for gastric surgery, or to help treat depression.

“To take the next step, we need new materials to be developed and we hope that will happen in the next two to three years.

“Not all conditions would respond to this technology but many would and these applications could reduce the need for drugs.

Instead of a person taking pills every day they could have a sensor fitted.

“What we are looking at will advance the field of prosthetics, provide enhanced function to prosthesis users and reduce the time involved to learn how to use the device because the movements will come naturally.

“We are 10-15 years away from these developments but we are already pushing the boundaries of the science.”

Dr Nazarpour believes that the technology does not need to stop at hands. Indeed, only 38 per cent of amputations in the UK annually are hands.

He said: “Because the basic theory is very similar we think it can work on people who have had legs amputated, that we can use the sensors to communicate with the brain to produce prosthetic limbs that respond more naturally.

“The technology will also have applications for patients with neurological conditions where reduced sensation is a factor.

“Some of the world’s major pharmaceutical companies are already investing significant amounts of money in researching the technology.”

However long it takes, the fact remains that bionics is changing more and more lives. Ted Varley, at Steeper, said: “This is an exciting field to work in. As a child I always wanted to work in the automotive industry and I went on to work for the likes of McLaren and Landrover but I would not go back to them now.

“If you design cars or televisions or mobile phones, each new one is pretty similar to the last one but every iteration we come up in prosthetics is pushing the boundaries.

“A new phone or television set will not change lives but our prosthetics are transforming lives and that is truly phenomenal.”

CONTINUED ON PAGE 62

61

BIOSCIENCE JOURNAL WINTER 2015

z FEATURE

CONTINUED FROM PAGE 61

Remarkable breakthroughs that herald the age of bionics He was one of the television hits of the 1970s, the Six Million Dollar Man with his super-enhanced vision and reconstructed bionic limbs that allowed him to run at incredible speeds and jump huge distances.

It was all science fiction but, four decades later, the fantasy is starting to become reality as science comes up with ever more advanced ways of tackling physical infirmity.

The Six Million Dollar Man was an American television series about a former astronaut, Steve Austin played by Lee Majors, who was given bionic implants after a terrible accident.

The show, based on Martin Caidin’s novel Cyborg, ran for five seasons from 1974 to 1978 and spawned several movies.

It was all very far-fetched but science has been taking the idea and developing its own version.

Take advances made in San Jose, California, in the United States as an example, where scientists have developed a system that aids those with sight loss (one of the Bionic Man’s enhancements was incredible vision).

The latest system, using contact lenses and eyeglasses, is controlled by the eyes: a wink of the right eye zooms in and a wink of the left eye zooms out.

The technology relies on contact lenses that contain tiny aluminum telescopes that interact with a pair of eyeglasses to move between normal and 3x magnification.

The telescopes were first developed with Defense Advanced Research Projects Agency funding as super-thin cameras for aerial drones but they were adapted as an aid for people with age-related macular degeneration, the loss of light receptors on the inner surface of the eye that blurs the centre of the visual field.

Now, optical engineer Eric Tremblay of the Swiss Federal Institute of Technology in Lausanne, Switzerland, has revealed a new accessory that may make the contact lenses more appealing for the average person by making them more reactive.

When a user covers one of the reflectors by winking, the glasses change their polarization.

Dr Kianoush Nazarpour Two kinds of polarized light take two different paths through the contact lenses, activating the normal or magnified view.

The research team, which includes the University of California, San Diego as well as experts at Paragon Vision Sciences, Innovega, Pacific Sciences and Engineering, and Rockwell Collins, described the system as ‘a huge leap’ forward for people with age-related macular degeneration, which is the leading cause of blindness among older adults in the Western world.

Meanwhile, work is under way in the UK on another facet of the Bionic Man, this time a hand that can sense pressure and temperature and transmit the information to the brain.

The £1.4m research project led by Newcastle University and involving experts from the universities of Leeds, Essex, Keele, Southampton and Imperial College London, aims to develop electronic devices that connect to the forearm neural networks to allow twoway communications with the brain.

Reminiscent, the team say, of Luke Skywalker’s artificial hand in Star Wars, the electrodes in the bionic limb would wrap around the nerve endings in the arm. This would mean that for the first time the hand could communicate directly with the brain, sending back information about temperature and pressure.

62

Funded by the Engineering and Physical Sciences Research Council, and led by Dr Kianoush Nazarpour, a lecturer in Biomedical Engineering at Newcastle University, the team hope to develop technologies to give amputees a limb that more closely mirrors the real thing.

Dr Nazarpour, who is part of Newcastle University’s Biomedical Engineering team, said: “The UK leads the way in the design of prosthetic limbs but until now one of the limiting factors has been the technology to allow the hand to communicate with the brain.

“If we can design a system that allows this two-way communication it would help people to naturally reach out and pick up a glass, for example, whilst maintaining eye contact in a conversation, or pick up an apple without bruising it.

“This will advance the field of prosthetics, provide enhanced function to prosthesis users, and also reduce the time involved to learn how to use the device because the movements will come naturally.

“The technology will also have applications for patients with neurological conditions where reduced sensation is a factor.”

Steve Austin it might not be but technology is well on the way towards breakthroughs that were just the stuff of fiction in 1970s.

Image ©Shutterstock, Inc & RoyaltyStockPhoto

Cell Line Development in CHO, NS0 & Sp2/0

with enhanced PQA assessment for biosimilar development

Antitope offers CHO, NS0 and Sp2/0 biosimilar cell line development programmes with bespoke quality assessment tailored to individual projects

Enhanced PQA assessment

Continuous assessment of product quality attributes (PQA) during cell line development helps ensure biosimilars retains the desired product characteristics and function, avoiding redevelopment costs and delays to the market. PQA assessment includes: • Product integrity • Product activity • Product aggregation • Product glycan profiling • QC testing

Delivering on expectations

Efficient production is key to biosimilar development and Antitope has developed technologies such as Composite CHOTM and pANT vectorsTM to ensure key cell line development requirements can be met, including: • High expression levels of your antibodies or proteins • Free of animal derived products throughout the process in a chemically defined medium • Management of transfer to a GMP facility for a smooth transition to scale-up • No downstream milestones or royalty payments

Visit www.antitope.com/biosimilar-cell-line-development