British Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform Meeting Royal Welsh College of Music & Drama Cardiff, Wales, United Kingdom Wednesday April 19-Friday April 21, 2017 Conference Abstracts.

Abstract

Human Gene TherapyVol. 28, No. 8 AbstractsFree AccessBritish Society for Gene and Cell Therapy Annual Conference and Joint UK Regenerative Medicine Platform MeetingRoyal Welsh College of Music & Drama Cardiff, Wales, United KingdomWednesday April 19–Friday April 21, 2017Conference AbstractsPublished Online:1 Aug 2017https://doi.org/10.1089/hum.2017.29044.abstractsAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Gene Therapy Against HIV: Fighting The Virus In DisguiseSubmission to BSGCT science writing competition By Bernadeta Dadonaite . PhD Student, University of OxfordThe world around you is teeming with pathogens. Lucky, your immune system is well trained to protect and guard you from any unwanted invaders. Pathogens, such as viruses, are in a permanent arms race with our immune system, constantly evolving to evade our defences. Perhaps the most successful pathogens are the ones which have learned to disguise their weapons. And what better disguise can there be than becoming a physical part of the target- just the kind of tactics used by HIV.HIV, or human immunodeficiency virus, is a pathogen, which infects humans and causes AIDS (acquired human immunodeficiency syndrome). Around 38M people worldwide are infected with HIV and, while with appropriate treatment patients can live for many years, there is still no cure or vaccine against the virus1. The major treatment for the people with AIDS is an antiretroviral therapy (ART). ART is essentially a cocktail of various drugs, which stop the virus at different stages in its life cycle. However, preventing the virus from replicating is only a part of the challenge, as HIV is very good at playing hide-and-seek. HIV primarily targets our immune cells- the very same cells which are meant to seek and destroy the invaders. When HIV encounters an immune cell it enters and becomes part of the cell by integrating into the cell’s genome. Integration into a host genome is an essential part of HIV’s life cycle and it is also a perfect Trojan horse strategy. When HIV becomes part of the cell’s genome it is no longer recognised as a foreign entity and can remain dormant there until the environment is safe to reveal itself. The ART drugs are only able to kill an actively replicating virus but the dormant virus can remain integrated into the genome for many years. Consequently, HIV patients have to use ART drugs every single day for the rest of their lives to prevent virus from re-emerging, killing the immune cells and leading to AIDS.The challenges for treating HIV infections still remain great, however, the incredible advancements in gene editing and therapy technologies are paving a way for a brighter future. Gene editing is basically an on-demand ability to change and modify any gene within a given genome. Because human genome has thousands of genes, the targeting of specific genes, without interfering with the functions of other genes, has always been a challenge. However, experiments using CRISPR (clustered regularly interspaced short palindromic repeats) technology (fig. 1) suggest that there is a way of eliminating the dormant form of HIV. The dormant HIV virus, also called a provirus, is essentially a gene in disguise. The cell’s surveillance system cannot distinguish the provirus from any other cellular gene. With the use of CRISPR, however, there is a possibility of cutting out this non-native gene from the genome (fig. 2). CRISPR can be specifically targeted to a single gene via a guide RNA molecule and the guide RNA can be made to recognise the HIV sequence in the genome. Once the CRISPR system has identified the site of the provirus, it can cut it out, which leaves the cell free of the HIV. While in laboratory experiments this strategy has been very successful2,3,4, the challenge has remained in being able to deliver the CRISPR system into a living organism. Recently, however, a combination of CRISPR and decades-long advancements in gene delivery methods showed the potential of this strategy in animals too. Adeno-associated virus vectors (AAVs) have for many years been used as treatment-delivery systems. AAVs can be made to carry almost any gene and target specific cell types. The genes carried by AAVs integrate into a genome and are expressed inside the cells like any normal gene. Last year the AAVs engineered to carry a provirus-targeting CRISPR have been used to remove an integrated HIV genome from mice5. As much as 90% of blood cells were successfully depleted of provirus in the mice, indicating the potential for the clinical use of this approach.Figure 1: How CRISPR worksFigure 2: Using CRISPR against HIVWhile gene therapy approaches to remove the provirus from a genome have still a long way to go, gene therapy methods that prevent the virus from entering a cell in the first place have already reached clinical trials. When HIV encounters an immune cell it has to bind a receptor on its surface. The receptor, called CCR5, acts like a lock into which HIV inserts its key and opens the door into the cell. Just like with any lock, a slight change in the CCR5 can prevent the key from fitting in. Interestingly, it has been observed that a small deletion in CCR5 gene can prevent or attenuate HIV infection. 1% of Caucasians naturally carry the CCR5 deletion, indicating that it is not deleterious for humans. This observation suggested that engineering patient cells to carry the CCR5 deletion could be a potential treatment against the HIV. Indeed, experiments in a lab using CRISPR to introduce the CCR5 deletion have successfully produced immune cells that are resistant to HIV infection6. First clinical trial exploring the same principles has also showed some promise. The patients whose blood cells where isolated and modified to carry CCR5 deletion showed a much slower re-emergence of the virus in their blood in the absence of ART7.Gene therapy has traditionally been used to treat genetic diseases, however, the advancements in gene editing technologies now enable the use of gene therapy to treat infectious diseases as well. Many challenges still remain for the use of CRISPR against HIV. HIV is renowned for its ability to change and evade many treatment strategies. Potentially, HIV could outsmart CRISPR by changing its sequence, so that CRISPR can no longer recognise it. Even more so, the use of CRISPR in humans has still many ethical and safety issues to be resolved. However, until effective vaccines against HIV are available, combination of ART and gene therapy approaches may be the only way of developing a cure against HIV.ReferencesRestocking our Cells’ Toolbox to Overcome DiseaseSubmission to BSGCT science writing competition By Lauren Davis. Undergraduate Student, University of ExeterHumans have been adjusting the characteristics of living things for thousands of years. We have our ancestors to thank for the breeding of juicier fruit, meatier cows and cuter dogs, amongst many others. Plants or animals with desirable feature were interbred, to create new and improved varieties, in a process known as selective breeding. It was not clear how selective breeding worked, until scientists discovered the ‘instruction manual’ behind all living things.The instruction manual they found was a long string of code, called DNA. Each living thing has a unique DNA code, a copy of which is stored in each cell (the tiny building blocks that make up animals and plants).The long ‘instruction manual’ is broken up into ‘chapters’, each one tells the cell how to produce a specific protein (the chemical tools the cell uses to do its job). These chapters are called genes, and they can be turned on or off in each cell. For example, skin cells and brain cells use different chapters of the instruction manual to do their job, turning on genes they need and turning off those they don’t.The discovery of this system can explain the phenomenon of selective breeding. When two animals or plants reproduce, half of each parent’s DNA is combined, mixing their characteristics together. Sometimes during this process, genes become damaged or lost. When an animal or plant develops without a copy of the correct gene, some of its cells can’t make the protien tools they need to do their job. This shows itself in humans as genetic disease- diseases that are caused by DNA changes and can be passed from generation to generation.Hemophilia B is a genetic disease where the Factor IX (Factor 9) gene is damaged. Usually Factor IX is made and released into the blood by cells in the liver. When an injury causess bleeding, Factor IX and other protiens help blood to stick together into clots, which lood flow. Since people with Hemophilia B have a damaged version of the Factor IX they produce little or no working Factor IX. This means their blood does not clot properly, so when they are injured, they can bleed for a dangerously long time.To treat Hemophilia B, Factor IX needs to be restored to functional levels in the patients blood, to allow for normal clotting. This can be achieved by protien replacement therapy, where working Factor IX protiens are injected into the patient regularly.As our understanding of DNA grows however, it is becoming possible to fix faulty genes, such as the Factor IX gene in Hemophilia B. For this, scientists took inspiration from viruses. Viruses are micro-organisms which enter human cells and infect them. They add their DNA to the DNA of the human cells, so that the human cell produces copies of the virus. Some viruses add their DNA in a seperate loop, whilst others add it into the human DNA code.Researchers are able to harness the ability of viruses to enter and infect human cells. Adding a copy of a corrected human gene into a virus, allows it to be transported into and used by human cells. This equips the cell with the protien tool it was lacking, causing disease. For example, the Factor IX gene was added into a virus that infects liver cells. As a result, when the virus was allowed to infect a human, it added its DNA (containing the Factor IX gene) to liver cells.In clinical trials the edited virus was infused into the blood of severe heamophilia B patients. All 10 of the men in the trial showed an increase in Factor IX production after the treatment, indicating that the virus had successfully reintroduced the correct Factor IX gene into liver cells.These results are promising for patients with genetic diseases like hemophilia B, as well as type I diabetes and cystic fibrosis which are some more common genetic diseases these techniques could be applied to. However, there are still some issues with the technology. When viruses do not combine their DNA with the human cell DNA, the replacement gene copy could get lost as the cells grow and divide, so the solution is not permenant. However when virsuses do combine their DNA into the human cell DNA, they do so randomly, which may damage other genes. These changes could make the patient vulnerable to cancer and other side effects.Therefore, the next step in this constantly developing area of science is the development techniques which can accurately add genes or cut genes from a specific place in DNA. This should allow for more efficient and targeted therapies for genetic diseases.Figure 1: DNA is the instruction manual behind all living things, divided into shorter chapters called genes. These genes each provide the instructions a cell needs to produce a specific chemical tool called a tool box (adapted from https://fragilex.org/fragile-x/genetics-and-inheritance/fmr1-gene/)Figure 2: Viruses are able to enter cells and add their DNA close to or within the human DNA, this means the cell is forced to produce copies of virus proteins, allowing more and more viruses to accumulate until the cell bursts and releases them. (adapted from Encyclopedia Britannica 2006)Figure 3: Viruses can be used to transport a correct version of the Factor IX gene in to human liver cells, allowing them to start producing the protien at a higher level and reducing the severity of patients disease (Adapted from http://www.innovationessence.com/gene-therapy/)Merry Christmas!Submission to BSGCT science writing competition By Gwilym Webb. PhD Student, University of BirminghamChristmas disease, until now, has been considered incurable— a disease for life.In the healthy, cut fingers and bruised blood vessels release chemicals, which then start chain reactions. These chemicals activate the first steps in sequences of protein machinery, with each then activating more in turn. Many amplified steps later, the last protein works to form the fibres of a clot and stem bleeding.But what if a link in this chain is missing or broken? In Christmas disease, the critical step provided by the enigmatically named protein Factor IX is lacking. A Christmas disease patient still has the right signals to start clotting, but lacks one of the protein messengers needed to complete the chain. As a result, a tiny nick or knock can cause dangerous bleeding.Christmas disease carries the name of the first known patient: Canadian Stephen Christmas. Like all others affected, Stephen bled far too easily. To reduce the risk of a dangerous bleed, he needed injections of Factor IX gathered from the blood of others. The missing protein needed replacing several times per week and despite this, each day there was the risk that he might still bleed. This could be a stroke if the bleeding was in his brain or, more likely, but agonisingly, into his joints after a tiny knock.But would it not be better if, instead of replacing the protein, the source of the protein could be replaced? This is indeed what a team from London have recently achieved. Their discovery came too late for Stephen Christmas, who died in 1993 from HIV transmitted by one of his many injections, but gives hope to many others who suffer the disease carrying his name.Each protein is manufactured by the cells of the body according to the many blueprints of the genes in our DNA. Most often, one gene creates one protein. In Christmas disease, the genetic blueprint for Factor IX has a mistake and the protein it makes does not form properly. So why not replace the gene?This is harder than it sounds. Imagine sneaking a few extra words into a manuscript that is repeatedly being copied out. And then this needs to be on a microscopic scale, thousands of times at once. And then it must escape the sentinel that is the immune system, which tries to destroy foreign genetic material.The solution came through hijacking an ape virus and taking advantage of its unique properties. The Trojan horse in question— adeno-associated virus 2— more commonly affects chimpanzees and gorillas but has several key points in its favour. First, the virus itself causes little illness: it is no good if the cure is worse than the disease. Second, because the virus homes in on liver cells, it naturally ends up in a part of the body only lightly patrolled by the immune system, even when injected in the back of a hand. A header of genetic code was also added to that for Factor IX. This header was only ever read— and translated into active protein— in the liver and so this ensures that the new Factor IX is only be produced there too. Third, the virus is known to particularly effective at mingling its genetic code with that of those it infects: to ‘copy and paste’ its message in. In this way, when the body makes proteins according to its original code, it makes the virus’ proteins too.So, the genetic code for Factor IX was first inserted into adeno-associated virus 2 and the new virus injected into volunteers. At first however, only a little Factor IX was produced. It seemed that body was recognising and attacking the virus and that the new code was not integrating well enough.Two more cunning tweaks were required. A second Trojan horse was employed: virus 2 was put into the outer coat of its cousin, virus 8. Fewer human immune systems recognise number 8 and so less is destroyed. Next, a second complementary code sequence for Factor IX gene was added to the virus. Not complimentary in a polite society sense, but the mirror code of the intended signal. The two copies bound together protecting each other until unravelling inside a liver cell.This new gene in a virus in another virus’ clothes was a tremendous success. When given to six people with Christmas disease, all needed far less Factor IX and several needed none: they were effectively cured. This meant fewer injections, less risk of catching unwanted viruses like HIV and, critically, less risk of bleeding.As well as helping Christmas disease patients, the most exciting aspect of this gene therapy technique is its potential use in other genetic diseases: cystic fibrosis, sickle cell anaemia and more are possible targets. However, further tweaking of the system is required to create these more complicated proteins.It might be that European royal families pay particular attention to these developments too: the genetic defect that causes Christmas disease runs through the (ex-)Russian, Greek and British monarchies, and was carried by Queen Victoria. Some will recognise Christmas disease better through its alternative name— haemophilia B— and their history lessons at school.ReferencesInvited SpeakersINV01 Gene edited T cell therapiesQasim WaseemProfessor11Great Ormond Street Institute of Child Health, UCL, London, United KingdomBSGCT Welcome and Symposium 1 (Clinical Breakthroughs), Concert Hall, April 19, 2017, 4:00 PM – 6:00 PMBiography:NIHR professor of cell and gene therapy and consultant in paediatric immunology at Great Ormond Street Hospital and Institute of Child Health, UCL. He is leading the development and translational application of gene engineered T cell therapies.Engineered T cell therapies have been amongst the first to incorporate gene editing strategies given the ability to harvest and manipulate cells ex-vivo. In an early clinical application, successful treatment of leukaemia was achieved using ‘off the shelf’ HLA mismatched T cells transduced with a lentiviral vector to express a chimeric antigen receptor against B cell antigen and simultaneously ‘edited’ using TALEN reagents to overcome HLA barriers. Disruption of the endogenous T cell receptor alpha chain constant chain locus was used to prevent alloreactivity, and multiplex targeting of CD52 conferred resistance to the lymphodepleting antibody Alemtuzumab. The initial applications have employed these cells in a limited manner, as a bridge to successful allogeneic transplantation. Wider applications of similar approaches are anticipated, both in malignant and non-malignant settings, with alternative CRISPR/Cas based editing following closely behind.INV02 Haemophilia gene therapy: where do we stand?Nathwani AmitProfessorBSGCT Welcome and Symposium 1 (Clinical Breakthroughs), Concert Hall, April 19, 2017, 4:00 PM – 6:00 PMBiography:Professor Amit Nathwani, a NIHR Senior Investigator, is the Director of the Katharine Dormandy Haemophilia Centre at the Royal Free Hospital and Professor of Haematology at UCL. He is the founder and CSO of Freeline Therapeutics. His academic group works on gene therapy for monogenetic disorders as well as cancer.In 2011, our group showed that it was possible to achieve therapeutic expression of coagulation factor IX (FIX) at between 1–5% in 10 severe haemophilia B patients following a single administration of a self-complementary, serotype 8 pseudotyped, adeno-associated viral (AAV) vector. The only toxicity observed was transient subclinical transaminitis at the high dose level, which resolved following corticosteroid treatment. FIX expression has remained stable in most patients for >5years permitting these patients to discontinuation of FIX prophylaxis without increasing the risk of spontaneous haemorrhage. We have not observed any late toxicities.In the last 5 years six new AAV-haemophilia B gene therapy trials have begun with the most promising data emerging from studies using the gain-of-function Padua mutation in the FIX gene. Two studies have report a 6-8-fold enhancement of FIX catalytic activity to 25–40%; levels that are approach normal FIX values. Further advance arse likely to emerge through engineering of capsids to improve the efficiency of AAV gene transfer to the human liver using substantially lower vector doses, thus further improving the safety profile of this vector. This should further improve safety, while easing pressure on.Progress has also been made with haemophilia A, a more challenging target for gene therapy. Using our codon optimised AAV-FVIII expression cassette a BioMarin sponsored study recently showed Factor VIII expression of between 12–271% in 7 severe haemophilia A patients recruited to the high dose cohort. Other gene therapy trial in haemophilia A are poised to begin in 2017.Therefore, rapid progress is being made in the field of haemophilia gene therapy. Attention has to now shift on vector production to improve efficiency, quality whilst reducing costs.INV03 Results and challenges of ex vivo gene therapy clinical trialsCavazzana MarinaDr11Biotherapy Department, Biotherapy Clinical Investigation Center, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris - Paris Descartes University, Paris France, Paris, FranceBSGCT Welcome and Symposium 1 (Clinical Breakthroughs), Concert Hall, April 19, 2017, 4:00 PM – 6:00 PMBiography:Marina Cavazzana (paediatrician, haematologist, Scientific Women of the Year 2012) focuses on studying the development of the immune system, genetic diseases of the haematopoietic system and cell/gene therapy. She has initiated several clinical trials based on the use of ex vivo gene modified cells to treat patients with inherited disorders.Over the last fifteen years, gene therapy has shown its powerful outcome to successfully treat genetic diseases such as X-linked severe combined immunodeficiency. Moreover, the results of several other trials have confirmed the clinical potential of gene therapy approaches in other settings such as Wiskott-Aldrich syndrome, and b-hemoglobinopathies where the bone marrow content of the different stem and precursor cells and the cells' relationship with the stroma have very specific characteristics.The optimization of gene therapy requires better characterization or identification of the features of bone marrow homeostasis in disease settings. Recent progress has been achieved in the harvesting and expansion of healthy hematopoietic stem and progenitor cells (HSPCs) but also in identifying the appropriate tools. The use of self-inactivated (SIN) retroviral vectors has significantly reduced the risk of insertional mutagenesis and has been the best choice for the introduction of a therapeutic gene into autologous HSPCs than first-generation gamma retroviral vectors.Although this progress is of great value, caution is required when translating these findings into a diseased HSPC setting: (i) in order to allow an engraftment of the corrected cells, gene-modified autologous HSPCs need a conditioning regimen that could be responsible for acute and chronic toxicity, (ii) cord blood cells do not have the same biological characteristics as their adult counterparts.Lastly, genome editing and homologous recombination technologies have undergone spectacular developments. In view of the impressive progress recently reported for the gene addition strategy, offering gene-editing approaches to patients affected by b-hemoglobinopathies would move gene therapy one step forward but would raise a number of ethical questions.INV05 Implementing a strategic approach for UK regenerative medicineBuckle RobJoint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AMBiography:Dr Rob Buckle is Chief Science Officer at the Medical Research Council (MRC), and Director of the £25m UK Regenerative Medicine Platform, a major national programme established by three UK research councils to deliver translational research that will drive new therapeutic approaches in regenerative medicine.Regenerative medicine is a dynamic field of research that holds the promise of providing new therapeutic approaches for a variety of conditions. To realise this goal there needs to be a concerted and interdisciplinary effort, encompassing academics, clinicians and commercial entities, and the promotion of a translational research agenda built on solid underlying science. Substantial progress has been made in the latter, while advances in biomaterial technology, gene therapy and manufacturing science provide optimism that impact in the clinical arena may be achievable in the not too distant future.The UK Regenerative Medicine Platform (UKRMP) is a £25m national programme, established jointly by BBSRC, EPSRC and MRC in 2013. It aims to promote translational research in the field and address the knowledge gaps and obstacles where more development is needed to underpin the delivery of new therapeutic approaches. The UKRMP involves five interdisciplinary research Hubs that bring together leading research teams from 17 universities. The UKRMP has linkage to 25 companies and operates in close cooperation with the Cell & Gene Therapy Catapult. It is also aligned with centres of excellence in regenerative medicine funded by MRC, WT, EPSRC and British Heart Foundation (BHF), and resources for the supply of high quality and ethically-sourced stem cell lines, the UK Stem Cell Bank and HipSci.This presentation will take stock of how the UKRMP has delivered against its original objectives, and will highlight plans for second phase investment in the Platform that are currently under development.INV06 BHF Regenerative Medicine CentresHarding SianProfessor11Imperial College London, London, United KingdomJoint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AMBiography:Sian Harding is Professor of Cardiac Pharmacology and Head of the Division of Cardiovascular Sciences at NHLI, Imperial College London as well as Director of the Imperial British Heart Foundation Cardiovascular Regenerative Medicine Centre. She is studying the pluripotent stem cell-derived cardiomyocyte, both for disease modelling and cardiac repair.There are three British Heart Foundation Centres of Regenerative Medicine led by Professors Sian Harding (Imperial/Nottingham); Andrew Baker (Edinburgh/Bristol/KCL) and Paul Riley (Oxford/Cambridge). They specialise respectively in Cardiac; Developmental Biology and Vascular, with many areas of overlap and collaboration, as well as partners from around the UK and abroad. This presentation will briefly describe their achievements since opening in 2013; their future strategy and opportunities for involvement.INV07 The British Society for Gene and Cell TherapyBaker AndyJoint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AMBiography:Andrew H Baker, BSc (Hons), PhD, FMedSci, FRSE is the British Heart Foundation Professor of Translational Cardiovascular Sciences and the Gustav Born Chair of Vascular Biology at the University of Edinburgh. He works on pathological vascular remodelling detailing the molecular and cellular cues that drive vascular damage. He is working on RNA-, cell- and gene-based approaches to target vascular damage to improve patient treatment and outcome. He is the outgoing President of the British Society for Gene and Cell Therapy.The British Society of Gene and Cell Therapy not only works to bring the scientific, clinical and commercial communities together for workshops and conferences, but represents UK-based gene and cell therapy in initiatives that are developing long term interests in key issues such as manufacturing.INV08 The MATCH Study – Autologous Macrophages for the Treatment of CirrhosisCampbell JohnProfessor11SNBTS Advanced Therapeutics, Edinburgh, United KingdomJoint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AMBiography:John Campbell completed his PhD in Pathology at Edinburgh in 1995 and has worked in the cellular therapy field for over 20 years in various academic and industry positions. He is national head of research for SNBTS with over 30 full time scientists working on cellular therapeutics in his groupMortality from cirrhosis has tripled in the UK over the last 3 decades. The only curative option for end-stage cirrhosis is liver transplantation, but demand for organs far outstrips availability. Therapies to regenerate liver function are therefore urgently needed. Prof. Stuart Forbes' group has carried out extensive research into the mechanisms of liver regeneration and have found that hepatic macrophages are important for both the reversal of liver fibrosis and the control of the differentiation of hepatocyte precursors to hepatocytes. Macrophages can be generated in large numbers from peripheral blood monocytes thus making them attractive for a cellular therapeutic approach. Collaborative work between SNBTS and the University of Edinburgh at SCRM has led to the development of a protocol to generate large numbers of macrophages from cirrhosis patients. Patients undergo steady-state leukapheresis, monocytes are isolated by CD14 CliniMACS Prodigy selection, and the monocytes are then cultured over 7 days to a specific macrophage phenotype. This work is the basis for the MATCH clinical trial (Macrophage Therapy for CirrHosis). In this presentation, I will detail the development of the cellular therapy product to full GMP standards to obtain MHRA licensure. I will highlight the challenges associated with manufacture and validation of a novel autologous cellular therapeutic from a heterogeneous patient population with significant disease. The