Abstract
Gene Therapy Against HIV: Fighting The Virus In Disguise
Submission to BSGCT science writing competition By Bernadeta Dadonaite . PhD Student, University of Oxford
The 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 virus 1 . 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 successful 2,3,4 , 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 mice 5 . 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.
How CRISPR works
Using CRISPR against HIV
While 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 infection 6 . 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 ART 7 .
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.
References
Restocking our Cells’ Toolbox to Overcome Disease
Submission to BSGCT science writing competition By Lauren Davis. Undergraduate Student, University of Exeter
Humans 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.
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
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)
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
Merry Christmas!
Submission to BSGCT science writing competition By Gwilym Webb. PhD Student, University of Birmingham
Christmas 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.
References
Invited Speakers
Great Ormond Street Institute of Child Health, UCL, London, United Kingdom
BSGCT Welcome and Symposium 1 (Clinical Breakthroughs), Concert Hall, April 19, 2017, 4:00 PM – 6:00 PM
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.
BSGCT Welcome and Symposium 1 (Clinical Breakthroughs), Concert Hall, April 19, 2017, 4:00 PM – 6:00 PM
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.
Results and challenges of ex vivo gene therapy clinical trials
Biotherapy Department, Biotherapy Clinical Investigation Center, Necker Children's Hospital, Assistance Publique-Hôpitaux de Paris - Paris Descartes University, Paris France, Paris, France
BSGCT Welcome and Symposium 1 (Clinical Breakthroughs), Concert Hall, April 19, 2017, 4:00 PM – 6:00 PM
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.
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AM
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.
BHF Regenerative Medicine Centres
Imperial College London, London, United Kingdom
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AM
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.
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AM
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.
The MATCH Study – Autologous Macrophages for the Treatment of Cirrhosis
SNBTS Advanced Therapeutics, Edinburgh, United Kingdom
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AM
Mortality 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 clinical trial is now moving through the dose escalation phase, and I will outline the product manufacturing results to date, as well as progression through the dosing regimen. Ref: Moore et al. Cytotherapy 2015 Nov;17(11):1604-16.
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AM
Recent clinical trial success of liver directed gene therapy for bleeding disorders raised hopes of potential cure for other liver cell based diseases. Development of AAV vectors underpin the successes so far and clinical translation in the near future. Challenges and opportunities of liver directed gene therapy as well the current strategies to treatment development will be discussed.
Selective vs widespread CNS gene transfer with viral vectors
Imperial College London, Brain Sciences Hammersmith Hospital Campus London, United Kingdom
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AM
We utilise lentiviral and Adeno-Associated Viral (AAV) based viral vectors to transduce CNS so as to model and treat incurable neurological diseases. Differential pseudotyping of lentiviral vectors and the use of alternate promoters led us to discover ways of biasing tropism to different neuronal cell types when applied directly into the brain. Robust localised versus more widespread neuronal expression was achieved depending of the viral envelope used with these vectors. We have utilised both types of pseudotyped lentiviral vectors for approaches in Parkinson's and Alzheimer's disease with the former having progressed through clinical testing. Surface engineering was also used to confer very restrictive specificity of transduction to specific motor neurons following intramuscular administration so as to treat models of motor neuron disease. Novel viral envelopes are still being explored for deriving lentiviral vectors with useful CNS tropisms.
Different AAV vector serotypes can be used to efficiently transduce brain through direct administration. AAV9 was the first serotype shown to be able to spread in CNS via intravascular and/or intrathecal administration. Directed evolution has recently produced vectors with improved transduction efficiency compared to AAV9 through these routes. We have explored these vectors for their enhanced efficiency and their potential for treating disperse neurological disease. In this talk we will overview these approaches and discuss pathways to clinical development.
Lund University and Skane University Hospital, Lund, Sweden
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 2), Concert Hall, April 20, 2017, 8:30 AM – 10:30 AM
Parkinson's disease (PD) is the second most common neurodegenerative disorder.
The progressive loss of mainly dopaminergic neurons leads to a dopamine deficit in the striatum and the cardinal motor symptoms: rigidity, bradykinesia and tremor. The disease can be sufficiently treated for several years with pharmacological dopamine replacement. As the disease progresses and dopaminergic neurons continue to be lost, these treatments become less effective and the disease becomes a burden for patients, their relatives and society.
Currently, there is no treatment available in clinical praxis to stop the disease from progressing or to reverse the symptoms. The relatively focal loss of mainly one type of neurons, however, makes PD a good subject for cell replacement therapies.
Studies in animal models of PD had shown that neuronal replacement and partial reconstruction of damaged neuronal circuitries is possible when using fetal dopaminergic neurons, and formed the basis for pioneering fetal cell transplantation trials in PD patients in 1987 in Lund, Sweden, and elsewhere.
The presentation will introduce the concept of cell replacement in PD, illustrate what has been achieved so far, introduce the current ongoing multicenter clinical trial with fetal dopaminergic cells (Transeuro) and give an outlook on future plans and challenges in the field of cell replacement for PD.
RJAH Orthopaedic Hospital & Keele University, Oswestry, United Kingdom
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 3), Concert Hall, April 20, 2017, 11:00 AM – 12:30 PM
Cell therapy has now been used globally for the repair of cartilage defects for two decades, with the main technique focussing on autologous chondrocyte implantation (ACI). Clinical outcomes are generally good for 80% of patients with those patients who benefit within the first 12 months' post-treatment, retaining clinical improvement for at least 16 years. Hence a biological treatment with cell implantation appears capable of avoiding osteoarthritis which commonly develops following cartilage injury.
The method of ACI has been modified via the use of alternative materials for a patch, or scaffolds, or using alternative cell populations, as well as simplifying the method of application. Mesenchymal stromal cells (MSCs) are increasingly being considered in place of chondrocytes; autologous and allogeneic MSCs have been used, sourced from different tissues and used both with and without culture expansion in vitro. Applying them via intra-articular injection reduces the cost and logistics present with standard ACI, with the use of allogeneic cells potentially simplifying the procedure further.
However, many clinicians unfortunately now find themselves denied the opportunity to treat their patients with these approaches due to the lack of availability of commercial licensed products and reimbursement issues of treatment costs. The challenging financial and regulatory issues need to be addressed for cell therapies to be commercially viable and be retained within the field of medicine. Cell therapy and regenerative medicine has shown promise in orthopaedics; let us build on it rather than lose the opportunity and potential lessons we can apply to other medical areas.
Saarland University Medical Center, Homburg/saar, Germany
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 3), Concert Hall, April 20, 2017, 11:00 AM – 12:30 PM
The adult articular cartilage has a very limited capacity for self-repair. Lesions resulting from trauma or in osteoarthritis cannot fully restore the original hyaline cartilage structure with mechanical integrity even though a number of clinical options have been developed to address this issue. Significant advances have been made in experimental and translational cartilage regenerative medicine using gene therapy and tissue engineering as independent technologies [1-5], yet there is little evidence showing the feasibility of combining such procedures to improve cartilage repair by temporarily and spatially defined expression of therapeutic candidate sequences in sites of injury. The highly effective recombinant adeno-associated viral (rAAV) vectors have emerged as the preferred gene delivery system for human gene therapy [1-5], allowing for the development of a number of clinical trials worldwide. Using tissue engineering platforms to deliver rAAV in a scaffold-derived manner is a novel approach to improve the current gene transfer systems to enhance chondroregeneration in the damaged cartilage tissue [6-10]. It may also circumvent some of the obstacles associated with the use of rAAV in the clinics among which the per-existing immune responses to the vectors in the recipients.
University of Oxford, Oxford, United Kingdom
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 3), Concert Hall, April 20, 2017, 11:00 AM – 12:30 PM
The lymphatic vasculature is a blind-ended network covering most tissues and organs of the body and is an essential component of vertebrate development and homeostasis. During pathological conditions, lymphatic vessels expand via lymphangiogenesis in order to aid the clearance of interstitial fluid and reduce inflammation. Whilst the response of the lymphatics is well documented at peripheral sites, there is minimal insight into their role during the pathophysiology of organ systems such as the heart. We investigated the cardiac lymphatic vessels following myocardial infarction (MI) and observed significant lymphangiogenesis, underpinned by reactivation of a developmental programme. VEGF-C treatment significantly augmented lymphangiogenesis resulting in improved cardiac function. To determine whether this effect might be mediated by an immunomodulatory role, we flow-sorted immune cells from VEGF-C treated hearts and observed increased clearance of macrophages to mediastinal lymph nodes by day 7 post-MI. The molecular phenotype of cleared versus retained macrophages was equivalent, suggesting a reduction in macrophage load in the heart alone was sufficient to correlate with improved outcome. Finally, we examined macrophage trafficking in two genetic mouse models with compromised lymphatic vessels: Lyve-1 knock-out mice which have impaired immune cell uptake and Prox1 haploinsufficient mice which have a restricted lymphangiogenic response to injury. In both cases, we observed significantly reduced macrophage clearance, and are currently testing whether this correlates with cardiac remodelling and function. These data suggest that invoking developmental lymphangiogenesis to modulate the innate immune response may represent a therapeutic target to promote optimal cardiac repair following injury.
Catalan Institute of Oncology, L'hospitalet De Llobregat, Spain
BSGCT Symposium 4 - Cancer gene and cell therapy, Concert Hall, April 20, 2017, 1:30 PM – 3:30 PM
Oncolytic viruses propagate selectively in tumor cells. Prospects for cancer virotherapy are improved by the ability of oncolytic viruses to induce antitumor immune responses. This has been demonstrated upon repeated intratumoral injection of an oncolytic Herpes Virus in cutaneous melanoma lesions. Adenoviruses have a unique life cycle and epithelial cell infectivity particularly suitable for oncolysis of solid tumors. However, lack of clinical efficacy indicate that oncolytic adenoviruses need to be improved at different levels, such as systemic tumor targeting, intratumoral spread, and the ability to induce anti-tumor immune responses. This talk will present strategies to overcome these limitations.
Kings College London, London, United Kingdom
BSGCT Symposium 4 - Cancer gene and cell therapy, Concert Hall, April 20, 2017, 1:30 PM – 3:30 PM
We have developed a chimeric antigen receptor (CAR) named T1E28z that targets the extended ErbB network. The T1E peptide is a promiscuous ErbB ligand that engages ErbB1 homo- and heterodimers and the ErbB2/3 heterodimer. CAR signalling is provided by a fused CD28 + CD3ζ endodomain. T1E28z is co-expressed using the SFG retroviral vector together with a chimeric cytokine receptor, 4αβ, that allows the selective ex vivo expansion of engineered T-cells using IL-4. Efficacy of the resultant “T4 immunotherapy” has been demonstrated in xenograft models of established head and neck, ovarian, breast cancer and mesothelioma in vivo, without significant accompanying toxicity. The CAR can also engage mouse ErbB receptors, enabling human T-cells to kill both mouse ErbB+ tumour cells and pulmonary endothelial cultures in vitro. Nonetheless, intravenous or intratumoural transfer of human T4+ T-cells into SCID Beige mice promotes tumour regression without clinical or histologically detectable toxicity. By contrast, administration of large doses of human T4+ T-cells using the intraperitoneal route elicits cytokine release syndrome, in a macrophage–dependent manner. These data demonstrate the existence of a therapeutic window for T4 immunotherapy in mice. To de-risk this approach in man, a phase 1 trial has been initiated in patients with locally advanced/ recurrent head and neck cancer in which intra-tumoural delivery is employed to minimize risk of toxicity. An update on the current status of the trial will be provided.
Barts Cancer Institute, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, EC1M 6BQ, London, United Kingdom
BSGCT Symposium 4 - Cancer gene and cell therapy, Concert Hall, April 20, 2017, 1:30 PM – 3:30 PM
Pancreatic adenocarcinomas have the lowest 5-year survival-rates of all cancers because of late presentation of symptoms and rapid development of resistance to current cytotoxic drugs. Late-stage metastatic prostate cancer is currently incurable. A promising approach to address the need for improved treatment strategies for these malignancies is the combination of cancer-selective oncolytic adenoviral mutants with current therapeutics.
Several adenoviral mutants have been evaluated in clinical trials with good safety profiles and enhanced efficacy in conjunction with cytotoxic agents. In preclinical models of pancreatic and prostate cancer, we demonstrated potent synergistic cancer cell killing with our engineered mutants in combination with DNA-damaging apoptosis-inducing cytotoxic drugs. The most potent mutant, AdΔΔ, has the small E1ACR2-region deleted, which binds to pRb, for selective propagation in cancer cells with deregulated cell cycle. In addition, the anti-apoptotic Bcl2-homologue E1B19K was deleted to increase the synergistic enhancement of apoptosis in combination with cytotoxic drugs such as gemcitabine. We found that AdΔΔ could act on multiple cellular pathways contributing to the reduced tumour progression in drug-insensitive cancer cells. We further modified the virus to express an αvβ6-specific peptide (FMDV) for tumour-targeting and incorporated an E3-gene deletion for arming with cytotoxic genes or immune factors.
We will present results with the combination strategy in 3-dimensional co-culture and in vivo models. Recent findings on how E1B19K-deleted adenoviral mutants can subvert the cellular defenses against viruses and cytotoxic agents will be discussed. By exploiting these mechanisms improved anti-cancer therapeutics may be developed to further enhance tumour cell killing.
New York Genome Center and NYU, New York, United States
BSGCT Symposium 5 - Genome Editing, Concert Hall, April 20, 2017, 4:00 PM – 6:00 PM
The easy programmability of CRISPR (clustered regularly interspaced short palindromic repeats)–associated nucleases like Cas9 suggests a new way to interrogate gene function at the DNA level instead of the transcript level. By combining genome editing with forward genetic screens, we have developed genome-wide libraries for negative and positive selection screening in vitro in human cells and in vivo in whole organisms such as mice.
In addition to genome-scale screens for protein-coding genes, we have developed techniques for adapting CRISPR screens into noncoding regions of the genome, where it can be challenging to identify functional elements. The noncoding genome plays a major role in gene regulation and disease yet we lack tools for rapid identification and manipulation of noncoding elements. We find that mutations at specific noncoding elements lead to changes in transcription factor occupancy and in the local epigenetic landscape. Changes in these noncoding regions are coincident with modulation of nearby gene expression. This expands the scope of pooled CRISPR screens to target the full genome — including both protein-coding and noncoding regions — for understanding fundamental genome biology, probing gene regulatory networks, and finding new mechanisms in drug resistance and therapeutic development.
Department of Physiology, BioSciences Institute, University College Cork, Cork, Ireland
BSGCT Symposium 5 - Genome Editing, Concert Hall, April 20, 2017, 4:00 PM – 6:00 PM
Small molecule therapies which target at least 12 of the 272 known disease-causing variants in the CFTR gene could ultimately provide an effective treatment for ∼97% of individuals with cystic fibrosis (CF). For those variants that cannot be treated by small molecules, gene editing by donor-dependent homology directed repair may be an option, however, efficiency is very low, and limited to dividing cells.
In contrast, targeted deletions by CRISPR-mediated non-homologous end joining (NHEJ) occur at a much higher frequency and do not require a donor sequence. We have identified at least three deep intronic splicing mutations in the cftr2.org database (1.2% of all alleles) that could potentially be repaired by CRISPR NHEJ. The variants 3849 + 10kB C > T and 1811 + 1.6kB A > G cause aberrant splicing of the CFTR mRNA due to the creation of cryptic donor sites that lead to the incorporation of a pseudo-exon and premature stop codons in the mature transcript. A third variant, 3272-26A>G creates a splice acceptor site which also results in a frameshift mutation and premature stop codon.
We have used CRISPR Cas9 and/or CRISPR Cpf1 to successfully create double-stranded breaks (DSBs) either side of these variants in the genome to remove short intronic sequences by NHEJ in a highly efficient manner (up to 70% of treated cells). Using plasmid based reporter assays, we have confirmed that deletion restores normal splicing patterns. We are currently evaluating this approach in primary cells derived from individuals with these variants, to determine the functional consequences of restorating normal CFTR splicing.
School of Biological Sciences, Royal Holloway, University of London, Egham, United Kingdom
BSGCT Symposium 6A - Gene and cell therapy for rare diseases, Concert Hall, April 21, 2017, 9:00 AM – 10:30 AM
The blood-brain barrier (BBB) is primarily composed of highly specialised brain microvascular endothelial cells (BMECs), together with pericytes and end processes of astrocytes. Although the BBB successfully maintains the brain microenvironment, it also blocks beneficial therapeutics for diseases of the central nervous system (CNS). To study the crossing of the BBB by potentially therapeutic agents, we have produced improved in vitro models using endothelial cells differentiated from human induced pluripotent stem (iPS) cells, following a published protocol 1,2. Three different clones of iPS cells were used, with positive results in all cases. The integrity of the BBB models was evaluated using trans-endothelial electrical resistance (TEER), expression of tight junction proteins claudin-5 and occluding, and permeability to para-cellular markers. The TEER of one of the iPS cell clones (∼7000 Ωcm2) was considerably higher than values reported for co-culture models (∼5000 Ωcm2)2. For comparison, the TEER of the most widely used in vitro model of the human BBB (hCMEC/D3 primary human brain endothelial cell line) is less than 50 Ωcm2. The permeability to fluorescent para-cellular markers (Lucifer yellow, LY, and sodium fluorescein, Na-F) was 40-fold and 3-fold less than in the hCMEC/D3 model, respectively. Our BBB cell models will have many potential uses including testing of therapeutic agents and investigating BBB breakdown in disease states.
1. Lippmann, E.S., et al., 2012. Nature Biotechnology. 30, 783-791.
2. Lippmann, E.S., et al., 2014. Scientific Reports. 4:4160.
This work was funded by The SMA Trust through the UK SMA Research Consortium.
Department of Haematology, University of Cambridge and NHS Blood and Transplant, Cambridge, UK, Cambridge, United Kingdom
BSGCT Symposium 6A - Gene and cell therapy for rare diseases, Concert Hall, April 21, 2017, 9:00 AM – 10:30 AM
Platelet transfusions to thrombocytopenic patients are increasing by 7–10% per year. We are currently entirely reliant on donor-derived platelets, which have limitations: short shelf-life and precarious supply chain, risk of donor-derived transmitted infections and issues of HLA mismatch in chronic recipients. We are aiming to develop protocols to produce platelets in vitro from a renewable source of stem cells - human pluripotent stem cells (hPSCs) - using a methodology and reagents compatible with the production of a clinical grade commercially viable product.
GlaxoSmithKline, Stevenage, United Kingdom
BSGCT Symposium 6B - Cell and Vector production, Richard Burton Theatre, April 21, 2017, 9:00 AM – 10:30 AM
Gene therapy using lentiviral vectors has been successfully demonstrated in the clinic for a growing number of conditions, but supply of clinical grade lentiviral vector at a scale appropriate to treat patient populations of hundreds or thousands of people remains a significant technical challenge. To date most clinical grade lentiviral vector manufacturing has used transient transfection of plasmid DNA into adherent 293T cells grown in cell factories. This manufacturing process is of limited use in large scale vector manufacture due to the high costs and long lead times involved in obtaining clinical grade plasmid DNA as well as the poor scalability of adherent cell factories beyond low 10s of litres. To meet the need for large scale vector manufacture GSK are establishing a cell line development process to generate stable lentiviral vector producer cell lines. The elimination of the transient transfection step during vector manufacture greatly simplifies the manufacturing process, improves its scalability and opens the possibility of lentiviral vector manufacture at the larger scales necessary for treatment of large patient populations.
&
Touchlight Genetics Ltd, Lower Sunbury Road, Hampton, TW12 2ER, United Kingdom
BSGCT Symposium 6B - Cell and Vector production, Richard Burton Theatre, April 21, 2017, 9:00 AM – 10:30 AM
We have exploited a unique process in which DNA can be amplified by a proprietary enzymatic process, developed by Touchlight Genetics, using rolling-circle amplification to produce DNA to commercial scale and quality. Configuration of the final product, rather than being circular, is closed linear double-stranded DNA (dbDNA™).
In this study, we aimed to produce lentivirus vectors using dbDNA™, and to monitor these vectors in vivo by incorporation of the luciferase transgene.
Large quantities of the vector backbone dbDNA™, consisting of an SFFV promoter driving a codon-optimised firefly luciferase, 2A cleavage sequence and GFP reporter transgenes were produced using this method and lentivirus vector was produced using these dbDNA™ constructs.
This vector was administered to neonatal CD1 mice via intracranial injection. Luciferase expression was quantified in conscious mice continually by whole body bioluminescent imaging. We observed long-term luciferase expression using this vector, which was compared to that observed with a plasmid derived lentivirus vector.
In conclusion, we are able to show that lentivirus vector can be produced using this novel dbDNA™ configuration. Since production is non-bacterial, a high level of DNA purity can be assured which is advantageous for clinical applications. Moreover, it may provide a unique platform to amplify problematic DNA and consequently produce vector for sequences that may prove difficult to manipulate in bacteria.
University of Sheffield, Western Bank Sheffield, United Kingdom
BSGCT Symposium 7A - Future challenges, Concert Hall, April 21, 2017, 11:00 AM – 12:30 PM
Human pluripotent stem cells (hPSCs) have the ability to self-renew indefinitely and differentiate into all types of tissue in the body, providing a potentially unlimited source of differentiated cell types for use in regenerative medicine, disease modelling and drug discovery. The use of hPSCs in these applications will necessitate the maintenance of large numbers of undifferentiated, genetically stable cells. Human PSCs are subject to mutations in vitro and in the presence of selection pressures, the variants with mutations that allow for improved growth outcompete their neighbours and overtake the culture. The commonly observed genetic changes in hPSCs are non-random and involve gains of (parts of) chromosomes 1, 12, 17 and 20, indicating that genes within these regions confer selective advantage to mutant cells. Genetic changes that arise in hPSCs during in vitro culture can affect their behaviour and confound experimental results. With hPSC derivatives entering the clinical trials, a possibility that genetic changes may confer malignant properties to hPSCs or their differentiated progeny is a major cause of regulatory concern. In our work, we are elucidating the molecular mechanisms that underlie the maintenance of the integrity of the hPSC genome, and how disruption of these mechanisms can lead to undesired genetic changes. We are also studying the functional effects of genetic changes on the behavior of hPSCs in vitro. Investigating the causes and consequences of genetic changes in hPSCs will help inform approaches to minimise their occurrence in hPSC cultures.
German Cancer Research Center (DKFZ)/National Center for Tumor Diseases (NCT), Heidelberg, Germany
BSGCT Symposium 7A - Future challenges, Concert Hall, April 21, 2017, 11:00 AM – 12:30 PM
The license of a recombinant (r)AAV-based gene therapy treatment in Europe and the exponentially increasing number of rAAV gene therapy clinical trials demonstrates these vectors' potential, though previous preclinical studies have dragged these vectors' genotoxic potential into the spotlight. rAAV genotoxic integration events were retrieved upon neonatal administration and from mouse models of human disease, which may indicate that the developmental stage at the time of vector administration as well as the target organ pathophysiology may affect eventual rAAV genotoxicity. However, the wealth of preclinical studies performed in both small and large animal models reporting the absence of rAAV-induced malignant transformation rather support rAAV to be a safe vector system. Moreover, rAAV integration studies performed on patient's biopsies further challenged the clinical relevance of rAAV genotoxicity as only genome-wide distributed integration sites could be retrieved and no adverse events associated with vector integration have been reported. The recently reported presence of wild-type AAV partial genomes in human liver cancer biopsies have further fueled the debate over rAAV gene therapy safety, particularly raising concerns over rAAV bearing wild-type AAV inverted terminal repeats. Taken together, these results highlight that the study of rAAV integration is critical to ensure gene therapy safety and that further studies are still needed to unravel the mechanisms involved in vector integration and eventual genotoxic effects.
BSGCT Symposium 7A - Future challenges, Concert Hall, April 21, 2017, 11:00 AM – 12:30 PM
The regulatory environment for the clinical trial of Gene and cell therapy products in Europe is multifaceted with the interplay of a number of regulatory areas. These include legislation covering the procurement of the starting material, through Gene Modified Organism requirements, product manufacture, clinical trial and licensure. This is compounded by the translation of many of these requirements differently by the various member states which can act to slow a multistate clinical trial. This presentation aims to detail the main areas of legislation, to highlight the areas of dissimilarity and to provide some practical advice to developers in the field.
Cell Therapy for Age-related Macular Degeneration
UCL Institute of Ophthalmology, London, United Kingdom
BSGCT Symposium 7B - Gene and cell therapy for blindness, Richard Burton Theatre, April 21, 2017, 11:00 AM – 12:30 PM
Age-related Macular Degeneration (AMD) is the leading cause of blindness in the developed world, with over 600,000 affected patients in the UK alone, and approximately 70,000 new diagnoses each year. There are currently no treatments available for the 90% of patients suffering with the dry form of AMD, which results in the gradual degeneration of the macular region over a number of years. AMD is associated with the deterioration of the retinal pigment epithelial (RPE), a supportive monolayer of cells that maintains the health of the overlying neural retinal cells. The loss of the RPE and subsequent degeneration of the photoreceptor cells leads to irreversible blindness. RPE cell replacement could be a potential therapeutic for AMD. The London Project to Cure Blindness has generated RPE cells from human embryonic stem cells and is investigating cell transplantation as a regenerative approach for AMD.
University College London,
BSGCT Symposium 7B - Gene and cell therapy for blindness, Richard Burton Theatre, April 21, 2017, 11:00 AM – 12:30 PM
Genetic eye disease is the commonest cause of sight impairment in children and adults of working age in the UK. Many gene defects involve the phototransduction cascade in rod- and/or cone-photoreceptor cells, and chromophore recycling in their supporting pigment epithelium, resulting in photoreceptor dysfunction and outer retinal degeneration. The eye has unique advantages as a target organ for gene therapy; local microsurgical delivery of recombinant adeno-associated virus vectors enables targeted sustained transgene expression in affected cells, with a degree of protection against systemic immune responses; the cellular architecture of the retina is readily accessible to high-resolution optical imaging, and its topographical function can be mapped by microperimetry and multifocal electroretinography. Clinical trials of adeno-associated virus-mediated gene therapy for childhood blindness owing to lack-of-function defects in RPE65 have demonstrated improvements in sight. Several other recessively inherited single-gene disorders of the retina are the subjects of clinical trials of gene replacement therapy. Vector-mediated expression of neurotrophic proteins is an alternative, generic approach to slow inherited degenerations independently of the specific gene defect. In conditions where degeneration of the outer retina is advanced, vector mediated expression of artificial, microbial or vertebrate opsins may confer a degree of light-sensitivity to surviving second- or third-order cells of the inner retina.
Fairbairn Award Presentations
T-cell Modulation Group, Cardiff University School of Medicine, Cardiff, United Kingdom
BSGCT Special Lecture & Fairbairn Award Presentations, Concert Hall, April 19, 2017, 6:30 PM – 8:15 PM
Adoptive transfer of T-cells genetically engineered to express a T-cell receptor (TCR) specific for a particular antigen is proving to be a promising avenue for cancer immunotherapy. However, one of the main limitations of TCR-transgenic T-cells is the presence of endogenous TCRs in the engineered cells that compete with transduced TCRs for surface expression and/or mispair with transduced TCR chains to create receptors with unknown, and potentially dangerous, specificities. We hypothesised that knockout of the endogenous TCR from human primary αβ T-cells by CRISPR/Cas9 system would result in a superior activity of simultaneously transduced TCR. As a model system, we used a phosphoantigen-reactive γδ TCR and an HLA-A2-restricted melanoma-specific αβ TCR. Importantly, T-cells transduced with both the TCR and CRISPR/Cas9 specific for endogenous TCR almost exclusively expressed the transduced TCR. The increase of TCR expression level in dual CRISPR/transduced T-cells resulted in a dramatic improvement of functional response to cognate cancer-associated antigens. The sensitivity to antigen increased up to 1000-fold, and majority of the dual transduced cells could express multiple antitumour cytokines and markers of cytotoxicity in response to tumour. Consequently, dual transduced T-cells lysed cancer cells more efficiently than T-cells transduced only with the TCR. Finally, we applied the TCR replacement system to investigate the antigen-specificity and antitumour reactivity of TCRs derived from tumour-infiltrating lymphocytes and peripheral blood of final stage metastatic melanoma patients who had successfully cleared the disease. Our results indicate that application of TCR replacement technology will be advantageous to both scientific and clinical applications.
Molecular and Cellular Immunology Section, UCL GOSH Institute of Child Health, London, United Kingdom
BSGCT Special Lecture & Fairbairn Award Presentations, Concert Hall, April 19, 2017, 6:30 PM – 8:15 PM
X-linked lymphoproliferative disease (XLP) arises from mutations in the SH2D1A gene encoding SAP, an intracellular adaptor protein predominately expressed in T cell lineages. SAP protein expression is fundamental for adequate immune responses mediated by cytotoxic T lymphocytes (CTLs) and T follicular helper cells (TFH). Clinical manifestations are characterised by haemophagocytic lymphohistiocytosis (HLH), lymphoma, dysgammaglobulinaemia and impaired humoral immunity. Curative treatment is limited to allogeneic haematopoietic stem cell transplant with outcome reliant on a good donor match. However, as the majority of symptoms arise from defective T cell function we investigated whether adoptive transfer of gene corrected T cells alone could be restorative. We observed that transplanting wild type CD3+ T lymphocytes into SAP−/− mice significantly improved humoral immunity back to wild type levels. Subsequently, we transduced CD3+ lymphocytes from SAP−/− mice with a gammaretroviral vector containing codon optimised human SAP cDNA before infusion into sub-lethally irradiated SAP−/− recipients. Animals were challenged 8–10 weeks' post-infusion with NP-CGG and analysis performed after 10 days. We demonstrated statistically significant improvement in germinal centre formation and NP-specific antibody responses with 20–40% engraftment of gene modified T cells. In parallel, using a SIN-lentiviral construct with codon optimised SAP transgene expression driven by the EFS promoter, we efficiently transduced T cells from XLP patients resulting in improved cytotoxicity and TFH cell function in vitro. Using an LCL lymphoma model in immunodeficient mice we have shown that transfer of gene corrected patient CTLs also reduces tumour burden. Overall this data supports the further development of an autologous gene corrected T cell approach to treat patients with XLP.
Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom
BSGCT Special Lecture & Fairbairn Award Presentations, Concert Hall, April 19, 2017, 6:30 PM – 8:15 PM
The clinical efficacy of adenovirus serotype 5 (Ad5)-based virotherapies is suboptimal due to its extensive off-target uptake, inactivation by pre-existing humoral immunity and lack of tumour-specificity. We have generated a luciferase-expressing, αvβ6 integrin-selective oncolytic Ad5.3D.A20 vector for stratified targeting to epithelial ovarian cancer (EOC). The triple combination of tropism-ablation mutations successfully blocked native interactions with coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins and coagulation factor 10 (FX). Furthermore, the fiber knob HI loop carries an inserted 20-amino acid peptide (A20; NAVPNLRGDLQVLAQKVART) that binds αvβ6 integrin with high affinity. αvβ6 is expressed in a third of all EOCs and a variety of other epithelial cancers, but is non-detectable in healthy epithelia.
Ad5.3D.A20 exclusively transduced αvβ6-positive EOC cell lines in vitro, and primary clinical ovarian ascites-derived EOC cultures ex vivo – even in the presence of highly neutralising malignant ovarian ascites. Furthermore, the vector showed favourable oncolytic activity in αvβ6+ cancer cells in vitro. The in vivo biodistribution profile of the replication-deficient version of the Ad5.3D.A20 vector following systemic delivery was assessed in non-tumour-bearing mice. Transduction of off-target organs – the liver, spleen, lungs, heart and ovaries – was significantly reduced by over 4 logs relative to Ad5, based on in vivo luminescence imaging. Additionally, we showed by qPCR that the vector genome copy number of Ad5.3D.A20 was 7 logs lower than of Ad5 in the livers.
Ad5.3D.A20 presents an exciting platform for advanced, intraperitoneally metastasised, αvβ6+ EOC, and has great potential for immuno-oncolytic purposes for a variety of αvβ6-expressing cancer types.
Department of Gene Therapy, National Heart and Lung Institute, Imperial College, London, United Kingdom
BSGCT Special Lecture & Fairbairn Award Presentations, Concert Hall, April 19, 2017, 6:30 PM – 8:15 PM
We have previously shown that a lentiviral vector (rSIV.F/HN) pseudotyped with the F and HN proteins from Sendai virus generates high levels of intracellular proteins after lung transduction. Here, we evaluate the use of rSIV.F/HN for production of secreted proteins for other disease indications. We assessed whether rSIV.F/HN transduction of the lung generates therapeutically relevant levels of secreted proteins in the lung and systemic circulation using α1-anti-trypsin (hAAT) and factor VIII (hFVIII) as respective exemplars.
Mice were transduced with rSIV.F/HN carrying either the secreted reporter gene Gaussia luciferase (GLux) or the hAAT or hFVIII cDNAs by nasal sniffing, and gene expression was determined in lung tissue, epithelial lining fluid (ELF) and blood up to 19 months after vector administration.
rSIV.F/HN-hAAT transduction lead to therapeutically relevant hAAT levels (70 μg/ml) in ELF, with stable expression persisting for at least 19 months from a single application. Secreted proteins produced in the lung were released into the circulation and stable expression was detectable in blood for at least 19 months. The levels of hFVIII in murine blood approached therapeutically relevant targets (∼1% of normal). rSIV.F/HN was also able to produce secreted hAAT and hFVIII in transduced human primary airway cells.
rSIV.F/HN transduction of the murine lungs leads to long-lasting and therapeutically relevant levels of secreted proteins in the lung and systemic circulation. These data broaden the potential use of this vector platform for a large range of disease indications.
Selected Oral Presentations
UK Cystic Fibrosis Gene Therapy Consortium, Edinburgh, Oxford, London,
BSGCT Welcome and Symposium 1 (Clinical Breakthroughs), Concert Hall, April 19, 2017, 4:00 PM – 6:00 PM
In preparation for a first-in-man lentivirus clinical trial in cystic fibrosis patients we assessed whether shedding of viable virus occurs after transduction of mice by nasal instillation. Mice were transduced with rSIV.F/HN expressing the sensitive reporter Gaussia luciferase (rSIV.F/HN-GLux) by nasal instillation (1e8 TU)/mouse representing an overage of ∼1000-23000 fold of the anticipated human dose) and immediately co-caged with untransduced animals. 7 days after transduction Glux was quantified in broncho-alveolar (BALF) and nasal (NaL) lavage fluid and lung tissue. There was no evidence of transfer of active virus particles. We next used a more sensitive in vitro assay (lower limit of detection: 0.2 TU/24-well). Mice were transduced with rSIV.F/HN-GLux by nasal instillation (1e7 TU/mouse, representing an overage of ∼100-2300 fold of the anticipated human dose) or remained untreated. Animals were culled 1 to 21 days after transduction (n = 6/group) and BALF (1:1 diluted in OptiMEM), NaL (1:1 dilution), serum (1:5 dilution) and urine (1:9 dilution), were placed onto A549 cells. After 24 and 48 hr the medium was changed and GLux expression quantified 72 hr after in vitro transduction. We did not detect active virus particles in urine and serum at any time-points, whereas active virus was detectable in NaL for 24 hrs (∼20-200 TU/150 ul sample) and in BALF for at least 3 weeks (20-200 TU/ 150 ul sample at 24 hrs and 2-20 TU/150 ul sample at later time-points). These data will contribute to performing risk assessments in the lead up to the trial.
University Of Manchester, Manchester, United Kingdom
BSGCT Welcome and Symposium 1 (Clinical Breakthroughs), Concert Hall, April 19, 2017, 4:00 PM – 6:00 PM
Mucopolysaccharidosis Type IIIB (MPSIIIB) is a paediatric, autosomal recessive lysosomal storage disease caused by deficiency in α-N-acetylglucosaminidase (NAGLU) enzyme. Absence of NAGLU leads to accumulation of partially degraded heparan sulphate (HS) in lysosomes, giving rise to cellular dysfunction. Affected individuals exhibit severe central nervous system degeneration with progressive cognitive impairment. Current treatments are ineffective.
We have developed a haematopoietic stem cell gene therapy approach (HSCGT) in a mouse model of MPSIIIB, synthesising a high-titre lentiviral vector driving the expression of NAGLU under the control of the myeloid-specific CD11b promotor (LV.CD11b.NAGLU). HSCs from autologous MPSIIIB animals were transduced with LV.CD11b.NAGLU and transplanted into myeloid ablated MPSIIIB mice, comparing disease outcomes at 6 months post-transplant to wild-type, untreated MPSIIIB and MPSIIIB receiving a wild-type bone marrow transplant. A dual treatment strategy combining the anti-inflammatory agent prednisolone with LV.CD11b.NAGLU was also tested.
NAGLU enzyme activity was substantially increased in the brains of LV.CD11b.NAGLU treated mice, indicating effective delivery of NAGLU enzyme via monocyte trafficking and engraftment. Treated mice exhibited correction of MPSIIIB behavioural phenotypes in open field tests, significant correction of brain astrogliosis and lysosomal compartment size and an accompanied normalisation of inflammatory cytokines. HS levels were also significantly reduced, with a concomitant reduction in HS sulphation. Interestingly prednisolone treatment alone reduced peripheral inflammation and corrected behaviour phenotypes, despite no obvious reduction in astrogliosis. Thus, although prednisolone cannot cure brain pathology, it can provide relief from neurological symptoms.
Here we present neurological disease correction of MPSIIIB mice by HSCGT, demonstrating proof of principle for clinical trial development to improve patient neurological function.
University Of Manchester, Manchester, United Kingdom
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 3), Concert Hall, April 20, 2017, 11:00 AM – 12:30 PM
Somatic cells can be de-differentiated towards pluripotency in vivo by ectopic expression of defined reprogramming transcription factors (Oct3/4, Sox2, Klf4 and cMyc, known as OSKM). Sustained expression of such cues triggers tumorigenesis, but strategies relying on transient in vivo reprogramming induce pluripotency and proliferation only temporarily, without teratoma formation. Here, we sought to achieve transient reprogramming within mouse skeletal muscle with a localized injection of naked plasmid DNA (pDNA) encoding OSKM. We hypothesized that such approach would enhance the regeneration capability of the tissue after a severe and clinically-relevant injury. A single intramuscular administration of OSKM pDNA rapidly triggered the expression of pluripotency genes (Nanog, Ecat1, Rex1) and a marker of myogenic progenitors (Pax3) in the healthy gastrocnemius muscle of various mouse strains, but significant levels were only maintained for 2–4 days. In agreement, distinct clusters of mononucleated cells expressing pluripotency and early-myogenesis markers appeared among the myofibers soon after injection, and proliferated only transiently. While no dysplastic lesions or teratomas were found, morphometric analysis suggested that the reprogrammed cells successfully re-integrated into the tissue. Nanog was also significantly upregulated in the gastrocnemius when OSKM pDNA was administered 7 days after surgical laceration of its medial head. Regeneration was enhanced in reprogrammed tissues, as shown by rapid appearance of centronucleated myofibers and significantly reduced fibrosis compared to untreated controls. While further studies are warranted to achieve optimal functional regeneration, these results demonstrate the potential of transient in vivo reprogramming as a novel gene therapy strategy for the treatment of clinically-relevant muscle injuries via generation of transiently-proliferative, pluripotent-like intermediate cells.
UCL Great Ormond Street Institute of Child Health, London, United Kingdom
Joint UK RMP & BSGCT Plenary Session (BSGCT Symposium 3), Concert Hall, April 20, 2017, 11:00 AM – 12:30 PM
Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genodermatosis caused by loss-of-function mutations in COL7A1, the gene encoding type VII collagen (C7), a protein central for anchoring fibrils (AFs) formation at the dermal-epidermal junction (DEJ).
Presently there are no curative treatments for RDEB but retrovirally transduced autologous epidermal grafts are being investigated and early safety studies of intradermal lentiviral engineered fibroblast injections are underway. An alternative approach aims to modify allogenic mesenchymal stem cells (MSCs) to provide a more generalized treatment for RDEB.
A third-generation SIN-LV vector encoding a codon optimized COL7A1 transgene under the control of the human PGK promoter was investigated for MSC modification. Transduction did not affect the phenotype with >97% of MSCs remaining positive for CD105, CD90 and CD73 markers and retaining their differentiation potential. Overexpression of C7 was confirmed by flow cytometry, immunofluorescence and Western blot in cell lysates and culture supernatants, indicating production and secretion of full-length C7. C7-MSCs were included in RDEB grafts for human:murine modelling experiments to assess basement membrane architecture. Histological assessment after 8 weeks revealed recovery of DEJ function with no signs of blister formation contrary to the grafts generated without C7-MSCs where severe blistering was observed. Species-specific antibodies and FISH analysis were used to confirm the human origin of the graft and the presence of MSCs, respectively. Importantly, detection of AFs by transmission electron microscopy provided evidence of structural recovery. Collectively these data demonstrate that C7-MSCs have the potential to rescue the disease phenotype in a preclinical model. Further experiments will investigate strategies for systemic delivery of C7-MSCs.
Institute Of Child Health, UCL, UK
BSGCT Symposium 4 - Cancer gene and cell therapy, Concert Hall, April 20, 2017, 1:30 PM – 3:30 PM
T cells transduced to express chimeric antigen receptors (CARs) against B cell and myeloid antigen targets are being investigated in early phase clinical trials. To date approaches to effectively target T cell malignancies have been hampered by the fundamental issue of ‘T on T’ cytotoxicity during manufacture of therapies where T lineage antigens are targeted. Improvements in gene-editing technology now offer the possibility of efficiently eliminating expression of such target antigens, thereby protecting CAR engineered cells from fratricidal effects.
We generated a lentiviral vector for delivery of a second-generation CAR incorporating a codon optimised anti-CD3e scfv (3CAR). Expression of endogenous cell surface CD3 was disrupted by editing the T cell receptor alpha chain locus in order to prevent expression and subsequent assembly of the multimeric TCR/CD3 complex. Consequentially, these cells escaped 3CAR cytotoxicity, and rapidly dominated cultures until almost all CD3/TCR expressing cells were depleted. The product was self-enriched for 3CAR+TCR-CD3- T cells without further processing. Specific cytotoxicity has was demonstrated against CD3+ leukaemic cell lines, and is being further investigated in human:murine chimeric models.
The 3CAR strategy provides a route to therapeutic applications against T-cell malignancies, although in vivo effects will by necessity have to be time-limited to allow reconstitution of normal CD3+ T cell immunity after eradication of leukaemia. This could be achieved by incorporation of suicide gene for drug mediated elimination, or by adopting a ‘bridging’ strategy to achieve leukaemic remission before a conditioned allogeneic stem cell transplant.
Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom
BSGCT Symposium 4 - Cancer gene and cell therapy, Concert Hall, April 20, 2017, 1:30 PM – 3:30 PM
Adenoviruses can be engineered to provide highly versatile, powerful replication-deficient or oncolytic agents for virotherapy. Selective replication and self-amplification in target cells gives oncolytic viruses a unique advantage; self-amplification at the point of need. While some 57 human adenovirus types have been classified across 7 species (A-G), pre-clinical development has historically focused on a single serotype: Ad5.
There is concern that the therapeutic efficacy of Ad5-based constructs may be constrained by pre-existing immunity to this common virus and limited expression of its receptor (hCAR) in the tumour microenvironment. We are therefore interested in Ad49 which is associated with naturally low rates of pre-existing immunity and efficient transduction in primary cells; these properties directly address the concerns regarding Ad5 based vectors.
To combine the well-characterised oncolytic potential of Ad5 with the wider transduction potential of Ad49 the whole fiber (F) or fiber-knob (Fkn) region were pseudotyped onto on the Ad5 using recombineering technology. The pseudotypes show both Ad5/49F and Ad5/49Fkn possess enhanced infectivity in models of lung carcinoma (A549 cells) and ovarian cancer (SKOV-3 cells) compared to Ad5. Moreover, the Ad49 knob region enables infection of cells that lacked expression of all recognised adenovirus receptors. We therefore suggest that Ad49 may utilise a novel primary receptor to promote entry.
Desktop Genetics Limited, London, United Kingdom
BSGCT Symposium 5 - Genome Editing, Concert Hall, April 20, 2017, 4:00 PM – 6:00 PM
Given the exponential use of CRISPR in research and therapeutic applications, there is a need to understand both intended and genome-wide outcomes of CRISPR experiments. Off-target editing events impact experimental outcomes by introducing double-stranded breaks in regions that may lead to genome instability, disruption of gene regulation, cell death or tumorigenesis.
We have developed a next-generation sequencing approach for investigating off-target effects of CRISPR genome-editing experiments. This approach involves the design of Tru-Seq Custom Amplicon (TSCA) primers for multiplexed target amplification and deep sequencing of predicted off-target sites.
In this study, the specificity of sgRNAs targeting the RAG2 gene were assessed in K562 myelogenous leukaemia cells and CD34+ human pluripotent stem cells. TSCA primers were designed targeting 125 predicted CRISPR editing sites. A total of 3000 amplicons were pooled for parallel deep sequencing on a single Mi-Seq run. FASTQ data was demultiplexed and predicted sites were analysed using a custom bioinformatics pipeline.
Results show limited cross-hybridization across TSCA primer pairs indicating high specificity to the target loci. Over 80% of amplicons are seen to achieve uniform coverage, negating target region bias across the 125 predicted sites. CRISPR editing events were detected at the predicted on-target site as well as several predicted off-target sites. Mutations in the population were detected to a sensitivity of 0.1% minor allele frequency.
These results present a novel approach for investigating genome-editing incidence rates and associated repair products. We suggest this approach as a massively scalable option for discovering, validating, and assessing the safety of genome-editing modifications compared to current assays.
UCL Great Ormond Street Institute of Child Health (ICH), London, United Kingdom
BSGCT Symposium 5 - Genome Editing, Concert Hall, April 20, 2017, 4:00 PM – 6:00 PM
Gene editing applications are reaching clinical phase testing, and initial applications have favoured T cell therapies where cells can be modified ex-vivo at high efficiency and then characterised before use. Examples include disruption of endogenous T cell receptor (TCR) to generate ‘universal’ allogeneic T cells, and targeting of PD1 expression to foster cell persistence and reduce exhaustion.
We have generated a novel self-inactivating lentiviral vector platform that couples therapeutic transgene expression with precise DNA scission effects through a hybrid terminal region which incorporates a Pol III promoter-CRISPR guide within a modified, enhancer-deleted U3 3’ long terminal repeat. Duplication of the CRISPRsg cassette following reverse transcription magnifies guide expression, and subsequent electroporation mediated delivery of Cas9 mRNA supports transient Cas9 mediated DNA cleavage. The ‘terminal’ CRISPR configuration has the advantage of supporting high titre vector production with no evidence of transgene interference effects. We have demonstrated efficient transduction of human T cells expressing a chimeric antigen receptor (CAR) and simultaneous disruption of TCR expression by CRISPR/Cas9 mediated disruption of the T cell receptor alpha constant chain, with over 80% of transduced cells becoming TCR-.
Further, magnetic bead mediated depletion of residual TCR+ cells yielded a final product that was >96% CAR+ and >99% TCR- with molecular signatures confirming on-target DNA non-homologous end-joining effects. In vitro cytoxicity studies and in vivo anti-leukaemic effects in humanised immunodeficient mice confirmed intact functional CAR responses. The platform is readily scalable and is being adopted for early phase clinical evaluation of anti-tumour cellular therapies.
Institute of Neuroscience and Psychology, University Of Glasgow, Glasgow, United Kingdom
BSGCT Symposium 6A - Gene and cell therapy for rare diseases, Concert Hall, April 21, 2017, 9:00 AM – 10:30 AM
Rett syndrome (RTT) is a neurological disorder characterized by severe impairment of motor and cognitive functions, primarily caused by loss-of-function mutations affecting MECP2. Using knockout and knock-in mouse models of RTT, we investigated the impact of vector design, dosage and delivery route on the efficacy and safety of gene augmentation therapy.
Systemic delivery of MECP2 to 4-5-week-old Mecp2-null mice using an AAV vector was performed. In this vector, the transgene was under control of a minimal endogenous promoter. Administration of this vector revealed a narrow therapeutic window. High doses resulted in liver toxicity whereas lower doses significantly extended the survival of Mecp2-null mice but had no impact on RTT-like neurological phenotypes. Similar results were observed after systemic delivery of the same vector to a Mecp2 knock-in model expressing a mutant T158M allele.
Modifications to the vector design were made, included the incorporation of an extended Mecp2 promoter and an endogenous 3’-UTR fragment. After systemic delivery of this vector to 4-5-week-old Mecp2-null mice, significant reduction in hepatic toxicity was observed.
To increase brain transduction efficiency, this modified vector was delivered to neonatal Mecp2-null mice via direct cerebroventricular injection. This resulted in increased survival and bodyweight, and an amelioration of RTT-like phenotypes. These results show that the ability to control levels of exogenous MeCP2 expression in the liver is achievable through modification of the gene therapy construct. They also suggest the importance of high brain transduction as a crucial factor to impact RTT-like phenotypes.
UCL School of Pharmacy, University College London, London, United Kingdom
BSGCT Symposium 6A - Gene and cell therapy for rare diseases, Concert Hall, April 21, 2017, 9:00 AM – 10:30 AM
Niemann-Pick Type C (NP-C) is a fatal lysosomal storage disorder with neurovisceral pathology, for which there is currently no major disease modifying treatment. In the majority of cases, loss of the late endosomal transmembrane NPC1 protein causes systemic intracellular lipid accumulation. Premature death is normally associated with progressive neurodegeneration and subsequent neurological disease manifestations. This project focuses on the development and pre-clinical evaluation of gene delivery to the brain in a well-characterised mouse model of NP-C using an adeno-associated viral (AAV) vector.
A single administration of an AAV9 vector expressing the human NPC1 cDNA via a neuron-selective promoter into the brains of neonatal wild-type mice resulted in successful over-expression of the human NPC1 protein. Supraphysiological expression of exogenous NPC1 was shown to not cause adverse effects in administered brains and produced NPC1 correctly localised to within late endosomal/lysosomal compartments. Administration in neonatal Npc1–/– mice resulted in a significant extension of average lifespan by over 120%, compared to untreated Npc1–/– mice (p < 0.0001). AAV-mediated gene therapy also prevented or ameliorated neurodegeneration in all monitored brain regions, resulting in the normalisation of various indices of motor function, along with significant improvements in weight and biochemical pathology. Additionally, the therapeutic efficacy of this gene therapy study was directly compared to several competing NP-C therapies. These proof of concept results demonstrate the use of gene therapy as a potential therapeutic option for clinical use in Niemann-Pick Type C and support the further development of this approach.
Loughborough University, Loughborough, United Kingdom
BSGCT Symposium 6B - Cell and Vector production, Richard Burton Theatre, April 21, 2017, 9:00 AM – 10:30 AM
Cell culture is a complex and dynamic process. Optimisation of cell quality and process economics, with defined and acceptable risk, requires an integrated approach comprising both experimentation and modelling. Dynamic mechanistic models offer the benefits of a mechanistic understanding of processes and can predict counter-intuitive outcomes over extended time periods. However, they are underutilised in the cell culture and bioprocess community partly due to the skills barrier to entry.
We have designed an accessible software interface for the description, testing and manipulation of hypothetical mechanistic dynamic models. The approach delivers parsimonious, and hence testable, models built on the dominant phenomena involved in cell culture (e.g. substrate-dependent growth, cell death or phenotypic commitment). We have applied the approach to determine the productivity limits of human erythroblast culture for user defined levels of risk and quality; the model enabled the effects of medium supply and cell density control on erythroblast growth to be predicted, and could optimise cells/cost production for any given facility time and medium volume cost. Relatively small shifts in strategy had multiple fold impact on outcomes and substantially changed the impact of imprecision in timing of process operations. A series of further case studies are under validation including a system model of hematopoietic stem cell growth dynamics with immobilised and soluble proteins, megakaryocyte production, and media supply influences on pluripotent stem cell growth dynamics. These models are not only powerful for in-silico optimisation of cell manufacture, but can generate insights into variability in research labs as well as new and testable hypotheses pertaining to mechanisms of culture development.
National Institute for Biological Standards and Control, South Mimms, Potters Bar, United Kingdom
BSGCT Symposium 6B - Cell and Vector production, Richard Burton Theatre, April 21, 2017, 9:00 AM – 10:30 AM
Lentiviral vectors (LVs) are widely used as gene delivery vehicles due to their ability to integrate their genome into both dividing and non-dividing cells. Currently, lentiviral vectors are usually pseudotyped with the glycoprotein-G of Vesicular Stomatitis Virus Indiana strain (VSVind.G). LVs with this envelope have several beneficial characteristics including broad vector tropism, physical stability and high titres. However, VSVind.G pseudotyped LVs can be inactivated by human serum complement which limits their in vivo potential. Therefore, scientists have been looking into alternative G proteins to pseudotype LVs. LVs that are produced using the G protein of Cocal virus (COCV), a close relative of VSVind, are reported to be resistant to inactivation by human serum complement (Trobridge, Mol Ther 18:725, 2010). In this study, we have confirmed the aforementioned findings relating to VSVind.G and COCV.G. We also investigated some mix-and-match chimeric constructs as well as various G proteins from other vesiculovirus species for their relative sensitivity to serum complement inactivation. Lastly, we expanded the study to additional sera from animals widely used in in vivo studies: rabbit, guinea pig, and mouse. We aim to further study the potential cell factors and G protein regions which might be involved in the inactivation processes and the species-specific inactivation responses.
Oxford University Hospitals Nhs Foundation Trust, Cancer Pharmacy Office, Level 2 Administration Centre, Oxford Cancer & Haematology Centre, Churchill Hospital, Headington, Oxford, OX3 7LE, United Kingdom
BSGCT Symposium 7A - Future challenges, Concert Hall, April 21, 2017, 11:00 AM – 12:30 PM
Brunel University London, Uxbridge, United Kingdom
BSGCT Symposium 7A - Future challenges, Concert Hall, April 21, 2017, 11:00 AM – 12:30 PM
A human IPS cell based in vitro model to evaluate the potential for gene therapy vector associated genotoxicity caused by retrovirus (RV) and lentivirus (LV) vectors would be useful as an alternative to the currently used in vivo models. Supported by the NC3Rs Phase I CRACK-IT Challenge initiative, we set up the basis of a standardized assay for an Individualized Genotoxicity testing (InGeTox) system to test both vector safety and efficacy. As the liver is recognised as the ‘gold standard’ for pharmacological toxicological evaluation, in this model, human induced pluripotent stem cells (hiPSc) are used for differentiation into long term 3D liver cell cultures whilst being subjected to gene delivery. This approach can use cells derived from the patient and enables the individual's genetic background to be accounted for in toxicological studies. In addition to a predictive test for integrative vector safety, we measured the effects of vector integration on gene expression. Aspects associated with vector integration that are suspected to influence gene expression include effects on gene promoters and enhancers, vector splicing with host genes to form aberrant transcripts and vector read-through. Also, initiation of host methylation on genes not associated with integration as a result of infection is investigated. We have created standards using known vectors that are either considered ‘safe’ or ‘unsafe’ to measure these effects to allow us to score the genotoxicity potential of new vectors that may be considered for gene therapy in the future.
UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, United Kingdom
BSGCT Symposium 7B - Gene and cell therapy for blindness, Richard Burton Theatre, April 21, 2017, 11:00 AM – 12:30 PM
The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage disorders characterised by neurodegeneration, early vision loss and premature death. A major obstacle to developing gene therapies is the challenge to efficiently deliver agents throughout the brain. Adeno-associated virus (AAV)-mediated gene therapies have been used for several photoreceptor degenerations to restore protein expression in photoreceptors and preserve retinal function. As loss of vision is often one of the first symptoms in NCL, severely affecting the quality of life of patients, we explored the therapeutic potential of an ocular AAV-mediated gene therapy in the Cln6nclf mouse, a NCL model deficient in the transmembrane protein Cln6.
We found that although Cln6nclf mice present with a predominant loss of photoreceptor function and photoreceptor cells; supplementation of CLN6 in photoreceptors is not therapeutic. The endogenous expression level of CLN6 is low in photoreceptors, yet high in bipolar cells, a cell type of the inner retina that is only lost in Cln6-deficient mice at late disease stages. We demonstrate that administration of the novel AAV serotype 7m8 results in an efficient transduction of bipolar cells. Surprisingly, the cell type-specific expression of CLN6 in bipolar cells slows significantly the loss of photoreceptor function and photoreceptor cells. Moreover, photoreceptors show a better cell morphology and Mueller glia cell activation was reduced following the bipolar cell-specific treatment.
This is the first study to show that the deficiency of a gene highly expressed in bipolar cells can cause photoreceptors to die and that a bipolar cell-directed treatment can slow the degeneration of photoreceptor cells.
Centre For Stem Cell Biology, University Of Sheffield, Sheffield, S10 2TN, United Kingdom
BSGCT Symposium 7B - Gene and cell therapy for blindness, Richard Burton Theatre, April 21, 2017, 11:00 AM – 12:30 PM
Deafness, a condition with a substantial social and economic impact, has no curative treatment. The best therapeutic option for profound hearing loss is the application of an electronic prosthesis, the cochlear implant. This device converts a sound stimulus into an electrical signal, functionally replacing the sensory hair cells. However, patients must have a working auditory nerve in order to convey the information to the brain. Thus, for many people, this is not a therapeutic option.
We have previously shown that hESC-derived otic neuroprogenitors (hONPs) can functionally restore the cochlear nerve in a gerbil model of auditory neuropathy. More recently, we have developed a new gerbil model with a two-pronged sensorineural hearing loss - auditory neuropathy is induced with topical ouabain and subsequently the hair cells are lesioned with a kanamycin/furosemide treatment. To recapitulate cochlear implantation, we are using a fully-implantable rodent stimulator in which the electrode is activated by a magnetic field. Initially, we implanted animals in which only the hair cells were damaged. Brainstem evoked responses were obtained after electrode stimulation, and animals showed behavioural changes compatible with auditory responses. We are now combining the cochlear implant prototypes with the rebuilding of the auditory nerve using hONPs. These progenitors were produced using a hESC reporter line, allowing for the purification of the otic cells prior to transplantation. Histology suggests that the transplanted cells survive and differentiate in the implanted animals, with neural fibres tracking towards the implant.
Session Speaker
Imperial College London, London, United Kingdom
UKRMP/BHF Session 1, Richard Burton Theatre, April 20, 2017, 1:30 PM – 3:30 PM
Tissue engineers strive to create biodegradable materials capable of directing tissue repair and regeneration; however, these scaffolds are often functionally inferior to native tissue. This is partly due to failure to fully recreate the complex ECM composition critical to tissue function. We are interested in creating biofunctionalized scaffolds with certain topographical or biochemical signals to stimulate growth and regeneration of new tissues. I will discuss three projects within the UKRMP Acellular Materials Hub that apply these techniques towards different applications. In the first project, we have targeted anti-fibrotic applications by functionalizing scaffolds with a novel laminin-111 β1 fragment. We discovered that this laminin fragment is capable of reducing the epithelial-to-mesenchymal transition in inflammatory environments and ultimately reduces the fibrotic response. In the second project, we have developed novel peptide-polymer conjugate systems, which specifically, non-covalently, and dynamically binds multiple glycosaminoglycans (GAGs) for the purpose of cartilage regeneration. We have investigated the chondrogenic effects of these GAG-binding peptides on human mesenchymal stem cells in biodegradable poly(ɛ-caprolactone) (PCL) scaffolds and collagen-mimetic hydrogels and foams. We have further developed methods for creating dual gradients of these peptides with a mineralizing peptide for guiding osteochondral regeneration. In the final project, we designed synthetic scaffolds with specific topographical cues to guide retinal repair. This work collectively investigates the development of biologically responsive functionalized scaffolds for anti-fibrotic, cartilage, and retinal applications in an effort to move toward biomaterials capable of recapitulating native ECM.
University/ BHF Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom
UKRMP/BHF Session 1, Richard Burton Theatre, April 20, 2017, 1:30 PM – 3:30 PM
Ischaemic heart disease is the leading cause of death worldwide. Myocardial regeneration after ischaemic injury requires the restoration of an efficient blood supply. Circulating endothelial progenitor cells (EPC) hold great promise as an autologous source of cells for vascular regeneration. However, at present, very little is understood regarding the mechanisms through which endogenous vascular cells contribute to neovasculogenesis post-MI. We investigated the hypothesis that vessel wall resident endothelial progenitor cells (EPC) drive endogenous vascular regeneration in the ischaemic heart through monoclonal expansion to form differentiated vascular progeny.
The Pdgfb-iCreERT2-Brainbow2.1 mouse has inducible expression of a green, red, cyan or yellow fluorophore in endothelial cells (EC). Fluorophores are inherited by EC progeny following division, permitting visualisation of the clonal architecture of the vessel wall during homeostasis and in regeneration post-MI. Clonal analysis of human tissues is possible through identification of patches of cells deficient in the mitochondrial enzyme, cytochrome c oxidase (CCO). Laser capture microdissection and Next Generation Sequencing of the mitochondrial genome of CCO deficient cells can confirm the clonal nature of the human blood vessel wall in health and disease, and validate the presence of a vessel wall progenitor cell niche. Our data show that EC in the human and mouse heart undergo clonal expansion, and that this is significantly upregulated in ischaemic disease. Evaluation of the mechanisms through which vessel wall EPC maintain vascular integrity will facilitate significant advances in regenerative medicine for the treatment of ischaemic disease.
University of Manchester, Manchester, United Kingdom
UKRMP/BHF Session 1, Richard Burton Theatre, April 20, 2017, 1:30 PM – 3:30 PM
Cartilage is an avascular tissue with limited ability to regenerate. Osteoarthritis resulting from cartilage injury and/or genetic mutations is a major untreatable cause of disability. Pluripotent stem cells (PSCs) provide an expandable source of cells with therapeutic and disease modelling potential. In order to develop new cartilage therapies and learn from cartilage diseases, our lab developed two methods to differentiate PSCs to chondrocytes.
1) The first recapitulates early development, resembling the condensing limb bud mesenchyme to generate PSC-chondroprogenitor aggregates in vitro (Oldershaw, 2010, Nat. Biotech.) expressing SOX9 SOX5/6, Collagen II XI and IX and Aggrecan but lacking Collagen X, a marker for hypertrophic chondrocytes. They generate hyaline-like cartilage in an osteochondral defect model (Cheng, 2014, Stem Cell Trans. Med.) reflecting potential for human articular cartilage repair.
2) The second method goes through an MSC-like intermediate and can model growth plate cartilage disorders e.g. multiple epiphyseal dysplasia (MED) in vitro. Patient-derived MED iPSCs (MATN3 p.Val194Asp mutation) and healthy control iPSCs were differentiated to an MSC-like intermediate, followed by chondrogenic pellet culture. Generated iPSC-MSCs display typical MSC morphology and markers (CD90, CD105, CD44 and CD73) and differentiate to bone (Alizarin red) and cartilage (SOX9, COL2A1, ACAN, BGN, DCN, MATN3 expression; Alcian blue positive pellets). COL10A1 and BIP expression were significantly higher in MED chondrogenic pellets, indicative of premature chondrocyte hypertrophy and ER stress respectively.
These models provide insight into PSC-differentiation and disease of cartilage and will enable screening of drugs for future treatment of both common and rare cartilage diseases.
University of Liverpool, Liverpool, United Kingdom
UKRMP/BHF Session 1, Richard Burton Theatre, April 20, 2017, 1:30 PM – 3:30 PM
Chronic organ disease or dysfunction is an increasing issue worldwide. Organ transplantation is an effective treatment but the pool of suitable organ donors is not sufficient to meet demands. Regenerative medicine therapies (RMTs) could decrease the need for transplantation in chronic injury by utilising exogenous cells to promote regeneration and reduce excessive fibrosis. Before RMTs can be used in man, their efficacy and biodistribution in preclinical models must be monitored.
MSOT has the imaging depth and fast scanning times required for dynamic imaging of pharmacokinetics and cellular biodistribution in mice. This study utilises MSOT to assess kidney and liver function in two murine injury models (adriamycin and CCl4 respectively) and to track distribution of labelled regenerative cells. IRDye800CW and ICG were used as contrast agents. In adriamycin-dosed mice IRDye took significantly longer to pass from the cortex to the pelvis of the kidney than in saline dosed mice; these data showed a positive correlation with the extent of histological damage. In the CCl4 dosed mice ICG clearance was imaged in the liver, gall bladder and in the blood and showed reduced clearance in the CCl4-dosed animals. To evaluate the ability of MSOT to monitor cellular biodistribution in real time, 10 million macrophages were labelled with gold nanorods, and administered to BALB/c SCID mice via the tail vein. The macrophages rapidly populated the liver, reaching 50% maximal engraftment within an hour.
This study demonstrates the utility of MSOT in quantitatively assessing organ function and cellular biodistribution.
Imperial College London, London, United Kingdom
UKRMP/BHF Session 1, Richard Burton Theatre, April 20, 2017, 1:30 PM – 3:30 PM
Cardiac cell therapy is the introduction of stem cells in the heart to repair/replace damaged myocardium. With current strategies, there is poor survival, integration and maturation of the injected cells, hindering the successful application of cell therapy in cardiology. Here we use myocardial slices to study the mechanisms of functional integration and electrical coupling of transplanted cells with the recipient myocardium.
Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were seeded and cultured in vitro on the surface of human and dog ventricular slices.
iPSC-CMs quickly attached to the slices, within 24 hours formed electrical connections with the other grafted cells and beat spontaneously. When the slices were cultured in unloaded condition, iPSC-CMs activity did not trigger the activation of the recipient tissue. When point stimulation was applied on a distant region of the slice, while the slice contracted, the signal did not propagate to the iPSC-CMs, suggesting a lack of coupling with the recipient tissue. At this time point however, myocardial slices cultured in unloaded conditions showed a significant degree of functional deterioration. Slice contractility decreased to 40% within 24hours. The application of mechanical load, in association with electrical stimulation, oxygenation and media circulation, could delay the deterioration of myocardial slices. iPSC-CMs could integrate more efficiently and they were able to respond to point stimulation, suggesting synchronization with the surrounding myocardium.
We conclude that integration of iPSC-CMs depends on myocardial structure and functional status, with important implications for the use of cell therapy in damaged areas of the myocardium.
Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology Medicine and Health, University of Manchester, UK
UKRMP/BHF Session 2, Richard Burton Theatre, April 20, 2017, 4:00 PM – 6:00 PM
Kidney failure is a devastating life threatening disease without cure. Development of human kidney tissue in vitro may open the way to cell therapies and also, through modelling disease, will enable us to identify new drug targets. We have shown that three different human pluripotent stem cell (hPSC) lines can reproducibly differentiate in culture to produce mini-kidneys, bearing a striking similarity to human kidneys in terms of cell type composition and morphology. We also constructed an array of lentiviral vectors expressing fluorescent and luminescent reporter genes and used them to transduce the hPSCs to allow us to trace the fate of cells and their differentiated derivatives in vivo. Transduction with a bicistronic iRFP-E2A-luciferase lentivirus resulted in robust, long-term expression of the transgenes without affecting the cells' pluripotent status or their capacity to differentiate. We then transplanted both labelled hPSCs and labelled differentiated kidney progenitors by subcutaneous injection into immunodeficient mice. The hPSCs retained their capacity to form teratomas and they were readily detected by in vivo bioluminescence imaging. The kidney progenitors were also detectable by bioluminescence imaging 12 weeks' post-transplantation. Moreover, they did not form teratomas, while morphological and immunostaining analyses on tissue excised from the injection sites showed that they formed structures encountered in the kidney in vivo. We have, therefore, developed technology for tracing hPSCs and their differentiated derivatives in vivo and demonstrated that the latter can form kidney tissue following transplantation, making a significant step towards their use for therapy.
Wellcome Trust - MRC Cambridge Stem Cell Institute, Cambridge, United Kingdom
UKRMP/BHF Session 2, Richard Burton Theatre, April 20, 2017, 4:00 PM – 6:00 PM
Parkinson's disease is a progressive neurodegenerative disorder with no current therapy that alters the course of the disease. The cardinal clinical manifestations of the disease – bradykinesia, tremor, rigidity and postural instability – are associated with the loss of dopaminergic neurons from the substantia nigra and can be ameliorated by the replacement of dopamine to the nigro-striatal system. The transplantation of dopaminergic precursor cells derived from fetal ventral mesencephalic tissue has demonstrated the potential of cell replacement strategies in Parkinson's disease, but has been limited by ethical and logistical constraints. An alternative source of cells for transplantation may be produced by differentiating pluripotent stem cells into dopaminergic precursors of the specific A9 midbrain subtype. Differentiation protocols that achieve this have now been successfully adapted to clinical grade reagents and culture systems. Pre-clinical testing to date has demonstrated the successful engraftment and function of these cells in animal models of the disease, without evidence of adverse effects such as tumour formation. Current issues being addressed include the cryopreservation of the cell product and the immunogenicity of the cells post implantation. Finally, validated assays establishing cell identity, potency and safety of the final product are in development as we move forward towards achieving regulatory approval for a clinical trial of this therapy.
The Jackson Laboratory, Bar Harbor, USA
UKRMP/BHF Session 2, Richard Burton Theatre, April 20, 2017, 4:00 PM – 6:00 PM
Loughborough Univerity, Center For Biological Engineering, Loughborough, United Kingdom
UKRMP/BHF Session 2, Richard Burton Theatre, April 20, 2017, 4:00 PM – 6:00 PM
Designing manufacturing processes to consistently produce process sensitive pluripotent stem cells (PSC) and cells derived from PSC of sufficient quantity and quality for clinical application is challenging and complex. The manual production of cell therapies in flask based processes is controlled primarily through adherence to SOPs which can allow variations in process by individual operators. This can lead to clinical production processes with little direct control of critical quality attributes, significant reliance on endpoint quality testing and subsequent high wastage costs, and overly large banking requirements. Demonstrating equivalence when manufacturing across multiple sites is required to ensure comparability and is considered “difficult for cell-based medicinal products”.
Researchers from the UK RMP PSC Platform (Cambridge, Sheffield and Loughborough Universities and NIBSC) are working with product and process developers including Lund University, I-Stem and Fraunhofer-IBMT to conduct scaled up experiments to: a. Understand the stability and variation of pluripotent stem cell cultures where different PSC lines are expanded and differentiated over multiple passages starting from independent vials. b. Demonstrate product equivalence when performing end-to-end production of a therapeutic from PSC where the production is done at the developer site, a second biological variation demonstrator site and the manufacturing protocol optimisation site. c. Automated expansion of PSC at three international sites emulating manufacture of cell therapies for global markets using the National Institute of Health “standard ruler cell line”.
The paper will report initial results of the application of a novel quality framework to permit manufacturing at multiple sites.
Selected Poster Presentations
Phage Therapy Group, Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Chemotherapy has been widely used for cancer treatment. However, chemotherapy does not selectively target only tumour cells and another major obstacle is the development of tumour drug resistance. To avoid these problems, the combination of chemotherapy with other therapeutic strategies has been used. Mammalian viruses are well recognised vehicles for gene therapy, but they are broad tropism. Our group has developed a hybrid vector from bacteriophage and adeno-associated virus (Phage-AAV), displays RGD4C peptide targeting specific receptor (αv integrin) on cancer cell and carries a transgene cassette for gene delivery. The Phage-AAV vector efficiently targeted, delivered, and expressed transgene in cancer cells. The vector gave the same result after intravenous injection of the vector in animal model of tumour. We used it as combination therapies with cancer drugs. Combination treatment of Phage-AAV vector carrying Herpes Simplex virus thymidine kinase gene (Phage-AAV-HSVtk) with doxorubicin increased the targeted cancer cell killing in 2D cultures and 3D tumour spheroids. We found that this increase in tumour cell killing was associated with a synergistic effect of doxorubicin on enhancing gene expression by Phage-AAV. We then combined Phage-AAV carrying transgene encoding short hairpin RNA to suppress mTOR gene expression (Phage-AAV-shmTOR) with temozolomide to treat medulloblastoma cells. The result exhibited that treatment of TMZ did not have effect on cell killing, but combination therapies of TMZ with the Phage-AAV-shmTOR significantly increased cell death. From the results above, we demonstrated that combination of Phage-AAV carrying therapeutic genes with cancer drugs is an effective strategy for cancer treatment.
Cardiff University, Cardiff, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
In ovarian cancer, resistance to chemotherapy represents a major clinical problem causing premature death. Oncolytic adenoviruses (OAds) are promising virotherapies that show synergistic effects when combined with chemotherapy agents, however clinical utility is commonly hampered by neutralisation by pre-existing antibodies in ascites. The molecular mechanisms of epigenetic aberrations and the capacity of OAds to induce cell death in ovarian cancer cells have not been studied.
Our data demonstrates up-regulation of HDAC2 in A2780/cp70 cisplatin-resistant cells compared to A2780 cisplatin-sensitive cells. OAd infected A2780 cells treated with cisplatin or the pan HDAC inhibitor trichostatin A (TSA) show increased cell death in comparison to OAd alone. Toxicity was enhanced by the combination treatment of cisplatin and TSA. However, whilst cisplatin and TSA enhanced OAd-mediated cell death in A2780 cells, OAd failed to achieve the same level of cell killing with that of cisplatin and TSA co-treatment, indicating an OAd-independent effect. The opposite was observed for A2780/cp70 cisplatin-resistant cells. OAd-mediated cell death was enhanced when cells were treated with TSA and a combination of cisplatin and TSA, an effect also observed when the cells were incubated in the presence of ascitic fluid. This suggests an OAd-dependent mechanism of cell death as uninfected cells treated with individual and combination treatments of cisplatin and TSA were refractory to cell death.
In summary, we demonstrate that TSA increases the sensitivity of platinum-resistant cells to oncolytic virotherapy in vitro and may offer an important combination strategy for improved outcomes for patients with late stage, chemotherapy-resistant ovarian cancer.
Cardiff University, Cardiff, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Adenoviruses (Ads) are commonly employed vectors for cancer virotherapy applications, both experimentally and clinically. The ability to visualise the progress of Ad infection as it spreads within a tumour would be useful in the development of more effective vector strategies. Protein IX (pIX) is a minor component of the Ad capsid and associates with the hexons that make up the facets of the icosahedron. In this study, we used recombineering technologies to generate Ad vectors, in which a fluorescent mCherry tag was fused to pIX to allow tracking of viral particles. We developed both replication deficient (E1 and E3 deleted), and oncolytic (delta24) Ads to enable the visualisation of replicating virus as it moves through tumour cells. Incorporation of a GFP transgene under the control of the HCMV promoter also enables visualisation of Ad transduction.
The mCherry tagged Ads could be propagated to high titres, indicating that incorporation of mCherry into the Ad capsid did not affect virus viability. After infection of SKOV3 (ovarian carcinoma) cells and HFCARs (fibroblasts over-expressing the native adenovirus receptor), fluorescent virus trafficking through the cell was observed. The virus was present around the cell membrane at early time points post infection, before trafficked through the cytosol towards the peri-nuclear region. At 24 hours, post-infection, GFP expression was observed indicating successful viral transduction.
In summary, we demonstrate fluorescent labelling of adenoviruses allows visualisation of cellular trafficking and spread between cells in real-time in vitro that can be used to develop efficacious adenoviral vectors.
CEINGE, Napoli
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Familial hypercholesterolemia is a genetic hyperlipidemia characterized by elevated concentration of plasma LDL cholesterol. Statins are not effective in all patients whose prognosis is still quite poor. In the past, we have developed safe and effective gene therapy strategies for the expression of anti-atherogenic proteins using PEGylated helper-dependent adenoviral (HD-Ad) vectors. We recently developed a HD-Ad vector for the expression of a secreted chimeric protein containing the extracellular portion of the human LDL receptor (LDLR) fused with a transferrin dimer. We evaluated the efficacy of LDLR-TF in CHOLDLA7, a cell line deficient for LDLR, in which we restored cell ability to uptake LDL; subsequently, we administered intravenously 1X10E13 vp/kg of the vector expressing LDLR-TF in LDLR-deficient mice and observed a reduction of total and LDL cholesterol and, consequently, of aortic atherosclerosis. Finally, we evaluated the biodistribution of this transgene with fluorescently-labelled LDL and observed accumulation in liver, intestine and heart after fluorescence molecular tomography analysis. These results constitute a proof of concept of the possibility of lowering LDL-C and reducing atherosclerosis using a secreted transgene and an alternative intracellular route.
Imperial College London, London, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Central South University, Changsha, China
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Although AAV-mediated approach is promising in haemophilia gene therapy, limitations of non-permanent expression, and risks of insertional mutation and immune responses remain. And such an approach is not applicable for haemophilia A (HA), mainly owing to the 7kb of the FVIII coding sequence that far exceeds the packaging capacity of AAV vectors. Nearly half of severe HA cases are caused by a 600kb inversion of F8 intron 22 (Inv22) that splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5′ part preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627bp coding sequence of exons 23-26. Here we describe an in situ genetic correction of Inv22 in patient-specific iPSCs. By using TALENs/TALENicases, the 627bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via HDR with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells derived from the gene-corrected iPSCs. This is the first proof of an efficient in situ genetic correction of the large mutation/gene via targeted addition of a small sequence. Differentiation and transplantation of the corrected endothelial progenitor cells into the animal models is ongoing for in vivo validation.
Cardiff University, Cardiff, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
L-selectin is an adhesion molecule expressed on naïve T cells, and proteolytically shed upon T cell activation. Despite activated cytotoxic T cells being routinely characterised as L-selectin negative, it has been demonstrated that T cells lacking L-selectin are not able to control solid tumour growth in an adoptive transfer model. We have previously shown that L-selectin is re-expressed on activated T cells after lymph node egress, and that activated T cells expressing non-shedding L-selectin are better able to home to sites of viral infection. It has also recently been suggested that down regulation of L-selectin on T cells by myeloid derived suppressor cells inhibits anti-cancer immunity through limited trafficking and antigen presentation. We therefore propose that T cells with non-shedding L-selectin will be better able to home to tumours, and, furthermore, that maintaining expression of this key adhesion molecule will improve the ability of transferred T cells to extravasate into the tumour stroma. Improving the ability of T cells to infiltrate solid tumours will help to expand the clinical applications of cell-based therapies such as genetically modified Chimeric Antigen Receptor (CAR) T cells, which are currently most often used to treat more accessible leukaemias, lymphomas and myelomas.
Great Ormond Street Institute of Child Health, London, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Umbilical cord blood (UCB) has unique and distinct properties, and harbours populations of entirely naïve T cells, suitable for manufacturing genetically engineered cell therapies. However, isolating and manipulating cord T cells using ficoll density gradient centrifugation is often challenging because of their highly heterogenous mononuclear cell compartment rich in nucleated red cells
CD62L (L-selectin) is an adhesion and homing molecule expressed on naïve T cells and is present on the majority of cord T cells. In three independent experiments, UCB units collected for research use by the Antony Nolan Cord Blood Bank were processed on the CliniMACS prodigy using CD62L microbeads to positively select T cells. CD62L selection successfully enriched the cord blood lymphocytes population from 30.2% ± 9% to 82.3% ± 6.5%. Crucially >85% of the enriched lymphocytes were CD3+ T cells, which were then activated using anti-CD3 and anti-CD28 Transact reagent and were efficiently transduced with a third-generation lentiviral vector encoding a chimeric antigen receptor. Transduction efficiencies were measured by flow cytometry and ranged between 27% to 58%. Over 9 days of culture CAR-UCB T cells expanded over 13-fold, and yielded >1x109 T cells from a single unit, meeting typical target expectations for trial dosing in adult recipients.
A robust automated manufacturing process to enrich and modify T cells from UCB has been established and forms a platform for allogeneic engineered cell therapy. The first clinical applications are likely to target relapsed leukaemia using CAR engineered cord T cells in the allogenic transplant setting.
Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Intrauterine haematopoietic cell transplantation (IUHCT) is a promising approach for pre-natal treatment of congenital genetic disorders. However, clinical trials for non-immunodeficiency disorders and preclinical models have reported limited success, with micro-chimerism and eventual loss of engraftment. We developed a murine model of thalassaemia major to study IUHCT followed by postnatal transplantation with immune-suppression.
Postnatal transplantation of bone marrow-derived mononuclear cells (MNCs) from adult mice were performed on IUHCT-treated pups that had received busulfan and fludarabine. This was because we suspected an immune-mediated mechanism of donor cell loss despite expectations of fetal immune naïveté at the time of IUHCT. Single and multiple postnatal transplantations were performed. Blood samples were collected from pups weekly for analysis through flow-assisted cytometry.
A single postnatal transplantation achieved higher but still transient increase in donor cell chimerism in pups that received a high-dose IUHCT (5E+6 MNC) as compared to low-dose recipients (2E+6 MNC). Low-dose IUHCT recipients that underwent postnatal transplantation sustained a longer interval of macro-chimerism (>1%) compared to controls which received only one dose in utero. Multiple postnatal transplantations effected sustained donor cell chimerism of >1% in chimeric pups, but not in non-chimeric pups. Immune-suppression prior to postnatal transplantation successfully sustained chimerism in non-chimeric subjects, with evidence of transient suppression of leucocyte counts. There is a significant increase in IgG in IUHCT-recipients before and after postnatal transplantations overall and specific to the strain of donor cell used; there is a non-significant increase in IgM levels in the same animals.
Further investigations of immune response in treated offspring following postnatal transplantation are required.
Central South University, Changsha, China
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Gene targeting could be stimulated strikingly by a DNA double strand break (DSB) created by engineered nucleases, while the DSBs are highly mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene targeting but with marked reduction of random mutagenesis/off targets. We previously demonstrated effective targeted gene addition at the human multi-copy ribosomal DNA (rDNA) locus, a genomic safe harbor for the therapeutic potential of transgene. To improve the transgene integration efficiency by using TALE nuclease while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickase) targeting this multi-copy locus. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus. Using TALENickases, the human FVIII gene with deletion of B domain (F8-BDD) was targted into the rDNA locus in haemophilia A (HA) patient-specific iPSCs. Both genetically modified iPSCs and derived endothelial cells expressed exogenous FVIII protein, indicating that transgene at the multi-copy rDNA locus of iPSCs holds a clinical potential for HA gene therapy. Interestingly, we found that more than one copy of the F8 gene was integrated into the rDNA locus, providing a more promising approach for not only haemophilia gene therapy but also genetic diseases involving multiple genes.
University Of Manchester, Manchester, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Graphene oxide (GO) has been shown to deliver small interfering RNA (siRNA) intracellularly, however as a component of more complex delivery systems mainly based on poorly biocompatible cationic polymers. Here, we explored the potential of GO as siRNA carrier on its own. The capacity of small GO flakes (lateral size <1 μm, obtained by the modified Hummer's method) to deliver siRNA was investigated in dividing and non-dividing primary cells and compared to that of a benchmark lipid-based transfection agent (Lipofectamine®).
GO complexed siRNA, against unfavorable electrostatic interactions, at GO:siRNA mass ratios (10:1, 20:1 and 50:1) that eluded cytotoxicity. Complexation protected the nucleic acid from degradation, even at 10:1 mass ratio and in the presence of high concentrations of nucleases that completely degraded free siRNA. A PCR-based method that allowed accurate quantification of intracellular siRNA was used to compare the delivery kinetics of both vectors. GO delivered siRNA rapidly (within 4 hours after transfection), but Lipofectamine® was able to sustain intracellular levels of the nucleic acid for longer and resulted in more efficient gene knock-down. Confocal microscopy to follow the intracellular trafficking of GO-siRNA complexes, where siRNA was coupled to nuclear targeting sequences, showed that the complexes were internalized and sequestrated in large vesicles while the nucleic acid was unable to reach its intracellular target. We provide previously unreported evidence that GO (without functionalization with polycationic groups) is able to act as a flat, 2-dimensional intracellular siRNA carrier, but conclude that further optimization of its transport and release capabilities will be essential to achieve effective gene downregulation
PPD, Granta Park, Cambridge, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Gene therapy is a rapidly developing field and, in recent years, an ever-growing number of gene therapy medicinal products (GTMPs) have advanced to clinical trials, and even to marketing authorisation stage. Regulatory frameworks in many countries have evolved rapidly to keep pace with the increasingly sophisticated demands of the pharmaceutical industry in the 21st century. Regulatory challenges faced by developers of GTMPs during early stage development will be discussed, ranging from the classification of borderline products to common issues encountered when applying for clinical trial authorizations (CTA) and genetically modified organisms (GMO) approvals. An overview of the early engagement opportunities that are available to developers of GTMPs will be provided, including classification procedures, scientific advice and pre-submission meetings.
University Of Cambridge/ Department of Haematology, Cambridge, UK
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Platelets are circulating anucleate blood cells produced by megakaryocytes (MK) in the bone marrow. Their primary role involves the formation of a platelet thrombus and support of the clotting cascade after vascular injury. Upon activation, platelets release various factors from α-granules, which promote their haemostatic function. Over the last decades, platelets have been recognised to act beyond these purely thrombotic processes and play crucial roles in a broad variety of (patho) physiological events such as tissue repair and angiogenesis through growth factors and chemokines contained in their α-granules.
We aim to use in vitro-produced platelets as vehicles for site-specific delivery of proteins with potential therapeutic effects. Our lab has developed a strategy, called Forward Programming to produce large quantities of MKs and platelets from human pluripotent stem cells (hPSCs) in GMP-compatible conditions. We first modified hPSC lines to eliminate the expression of HLA-class I molecules and generated universal platelets for future transfusion medicine. Second, using the granule-targeting moiety (SPD2-domain) of the Von Willebrand Factor (vWF), we have demonstrated the efficient expression and localisation of fusion reporter proteins (e.g. GFP) into the α-granules of forward programmed MKs and their platelet progeny. We are now focusing on the packaging of candidates, such as VEGF121, bFGF and CXCL2 which could potentially lead to improvement of revascularisation of ischaemic tissues and/or reduction of inflammation.
By genetic engineering of hPSC lines we seek production of customised universally compatible platelets loaded with proteins of huge therapeutic potential in regenerative medicine.
UCL, London, UK
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Early development of mammalian embryos occurs in a relatively low oxygen microenvironment in the reproductive tract (1.5- 5.3% O2). Yet, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are routinely cultured in atmospheric conditions. In this study, our aim was to investigate the effect of different oxygen tensions on the short-term culture of human iPSCs and on stem cell-fate determination during early differentiation. We performed gene-profiling analysis of human iPSCs maintained under normoxic (20% O2) and a range of hypoxic (0%, 2%, 5% and 8% O2) conditions. The expression of genes associated with pluripotency, embryonic germ layers and hypoxia were studied using qualitative RT-PCR, Immunostaining and Flow Cytometry. Preliminary results revealed that after four days of culturing human iPSCs at different hypoxic levels, morphological changes were observed. Additionally, hypoxia down-regulated the expression of pluripotency markers. Hypoxic conditions also promoted the expression of genes associated with the three germ layers and genes that are involved in the hypoxia-signalling pathway. Interestingly, mild hypoxia (8% O2) increased the number of cells expressing Brachyury (Mesodermal marker), while acute hypoxia (0%-2% O2) caused human iPSCs to differentiate into ectodermal lineage indicated by Nestin up-regulation. Thus, our results suggest that hypoxia is an important component of in vitro differentiation for the generation of clinically relevant progenitors.
Department of biomedical science, The University of Sheffield, Sheffield, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Dental pulp cells (DPCs) have been widely described as cells with mesenchymal stem cell characteristics. However, there is evidence suggesting the presence of a NCSC population as well as neurogenic populations within dental pulp cultures. It has been proposed that due to their ectodermal origin, hDPCs could be prone to commit to a neuronal or glial cell fate. Nevertheless, hDPCs in vitro are rather heterogeneous, which can affect the efficiency of differentiation as well as hampering possible therapeutic applications.
Thus, our aim is to identify and enrich for a subpopulation of hDPCs with neural crest-like or neural progenitor characteristics. Our intentions are to use these cells to generate auditory sensory neurons and Schwann cells for potential applications into hearing loss and peripheral nerve injury therapies, respectively.
Our experimental approach consisted of testing different culture conditions that would favour the growth and expansion of neurogenic cells. Additionally, we attempted a direct isolation of a P75+ve subpopulation by FACS. Cells were characterised by ICC and qPCR and their differentiation potential to neurons and glial cells was tested.
Our initial results indicate the presence of cells expressing markers related to neural crest and neural progenitor cells. We also identified a small P75 fraction of hDPCs. Differentiation didn't result in a clear sensory neural cell fate or a glial phenotype.
This would suggest that hDPCs, although neurogenic, require a complex interaction of factors to differentiate into sensory neurons. The interaction between DPCs and cochlear hair cells is currently being explored in culture explants ex vivo.
The University of Sheffield, Sheffield, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
We describe the generation of a three-dimensional culture of human intestinal tissue, derived from induced pluripotent stem cells. Our current work has focused on modifying and optimizing existing protocols for the generation of human intestinal organoids. We have examined gene expression patterns and used light sheet microscopy to characterise our protocol. The definitive endoderm protocol makes cells which are positive for SOX17, FOXA2, BMI1, LGR5, HES1, and VIM. Analysis of the hindgut stage indicates a presence of CDX2, FOXA2, BMI1, LGR5, HES1, VIM, and VIL1, suggesting the presence of progenitors for all the main cell types of an intestinal organoid. The organoids show the formation of hollow, spherical structures, where the inner layer is comprised of CDX2+ cells, and the outer layer is COL1A1+, suggesting the presence of an intestinal epithelium supported by a mesenchyme. Gene expression analysis revealed an increased expression of BMI1, ASCL1, VIM, HES1, VIL1 and, CDH1; these last three markers, together, indicate the formation of an absorptive epithelial barrier. Conversely the expression levels of LGR5, ATOH1, CHGA, DEFA5, and MUC2 was globally decreased, indicating a potential decay in the number of dividing intestinal stem cells, and suggesting the absence of a representative secretory lineage within the organoid. The upcoming experiments will address further modifications for the long-term expansion and maintenance of the organoids, so as to increase the secretory lineage population. Finally, we plan to develop a model of HIOs, compatible with the demands of high-throughput screening.
Centre for Stem Cell Biology and Department of Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Damage to the cochlear spiral ganglion neurons leads to irreversible deafness in a condition known as auditory neuropathy. Our laboratory has developed protocols to generate otic progenitors from human pluripotent cell lines, and established the proof of concept that these cells can be used to repair the damaged auditory nerve in an animal model. To translate these developments into a clinical application we aim to adapt the research-grade protocols currently in use into GMP compliance.
Existing research protocols use pluripotent cell lines cultured on mouse embryonic fibroblasts (MEFs). The process of differentiation involves dissociation with porcine trypsin and seeding on mouse laminin-1. In an attempt to adapt the initial phases of the method, we aimed to replace these reagents of animal origin with xeno-free counterparts; recombinant human laminins and dissociation by EDTA.
EUTCD-grade MasterShef lines, grown on a feeder-free system or on human fibroblast have been compared to the same MasterShef lines grown on MEFs. Otic differentiation was assessed by the formation of progenitor colonies and the expression of otic markers. While all the MasterShef lines tested are capable of otic differentiation, it would seem that the process is less efficient if the cells have been maintained feeder-free. Early results also suggest that cells coming from a feeder free culture do not differentiate well on mouse laminin. We are exploring different combinations of human recombinant laminins to optimise the substrate that would better support differentiation.
University Of Sheffield, Sheffield, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
In vitro protocols to generate progenitors of a particular lineage from human pluripotent stem cells are notoriously inefficient, leading to low cell yields. We have previously employed ligands crucial for otic placode induction, FGF3 and FGF10, to generate otic progenitors from human pluripotent stem cells. This protocol yields 15–20% of otic progenitors expressing high levels of key otic progenitor markers; PAX2, PAX8, SOX2 and FOXG1. Canonical Wnt signalling has been shown to play important roles in early gastrulation events and also in the expansion of the otic placode in vivo, and thus was manipulated in combination with the FGF ligands to investigate the effect on otic progenitor differentiation in vitro. It was found that an initial period of canonical Wnt signalling inhibition in combination with FGF3 and FGF10 promotes an ectodermal identity, with a concomitant increase in the expression of the otic markers. Following this, a phase of canonical Wnt activation further expanded the proportion of otic progenitors expressing high levels of the key markers to approximately 40–60%, substantially more so than in the previously established protocol. The progenitors derived from this more efficient, developmentally informed protocol also possess the ability to further differentiate into the more mature hair cell-like cells, and in particular, auditory neurons.
Regenerative medicine is emerging as a potential option for the restoration and repair of lost or damaged cells in the inner ear. Increasing the yield of otic progenitors differentiated from human pluripotent stem cells is crucial for a cell-based therapy for hearing loss disorders to become a reality.
Centre for Stem Cell Biology, University of Sheffield, Sheffield, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
The progression of stem cell-derived products from lab to clinic is dependent on the reliable purification of specific cells from heterogeneous populations. Various methods for differentiating pluripotent cells along lineages associated with the developing auditory system have been reported; hence it is important to devise mechanisms to isolate the cells with potential to treat hearing impairment.
A cell surface antibody screen has been undertaken on otic progenitors generated from human embryonic stem cells. Screen plates were imaged by automated microscopy thereby maintaining information relating to cell and colony morphologies. This information was used to narrow down potential hits based on known characteristics of otic progenitors. Five antibodies have been taken forward to verify their usefulness for isolating key progenitor subpopulations from diverse cell cultures via fluorescence-activated cell sorting (FACS). These include antibodies that would define different fractions of the progenitor population, potentially enabling the isolation of cells biased to differentiate towards either sensory neuronal or inner ear hair cell fates, both lineages being of interest in the restoration of hearing. A further antibody has been trialled as a means of marking non-progenitor cells for removal.
Early experiments indicate that the selected antibodies can be used in combinations to isolate progenitor subpopulations. It is hoped that accurate isolation of auditory progenitors from stem cell derived populations will aid progression from in vitro discoveries to therapies for deafness.
Obstetric & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
In utero hematopoietic stem cell transplantation (IUHSCT), a non-myeloablative approach for prenatal treatment of a variety of congenital hematological and immunological disorders, may serve as an alternative option to mainstream treatment of congenital haemoglobinopathies, with a small cell dose given to the fetus during the period of pre-immunity that may affect a greater engraftment and minimize the risk of graft versus host disease (GVHD) at the same time. We have designed a non-human primate (NHP) model of IUHSCT by injecting maternal bone marrow-derived mononuclear cells (MNCs) into the fetus to determine donor cell engraftment and the feasibility of this approach.
Fetuses (n = 10) received 18.9-170 × 106 MNCs (median dosage 49.8 × 106) labeled with carboxyfluorescein succinimidyl ester (CFSE). MNCs were delivered fetoscopically by injecting into the umbilical cord or, by ultrasound-guided transabdominal intracardiac injection, at 0.46-0.74 gestational age (71-144 days). Fetuses were then sacrificed at 24–48 hours post-injection. Fetal weights ranged from 36-160g. Targeted organs were harvested from fetus for analysis through stereoscopy, flow-assisted cytometry (FACS) and qPCR of MHC polymorphisms.
Stereoscopy results reveal detectable levels of maternal MNCs in fetal liver, spleen, heart, lung and placenta, which corresponds to FACS results. Low levels of maternal MNCs were found in haematopoietic tissues (fetal liver, spleen and heart, 2–8%). However, qPCR results indicated maternal MNCs were mostly found in placenta (6.9%), while other organs were reported to have relatively low levels of chimerism (<0.01%).
It is challenging to consistently deliver cells via fetoscopy, and time-consuming. Future studies on feasible options such as intra-cardiac or intra-peritoneal cell deposit is required.
The Cell and Gene Therapy Catapult, 12th Floor Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
The mechanisms to maintain pluripotency during expansion of pluripotent stem cells (PSCs) to date remains challenging and is associated with varying levels of spontaneous differentiation as well as genomic aberrations. Any changes to the quality of the cells have to be detected in order to pass or fail produced batches of cells. Differentiation of pluripotent cells can be detected by gene expression analysis, where reduction of expression of self-renewal genes and increased levels of expression of differentiation markers is observed.
Current methods for gene expression analysis such as hPSC Scorecards™, can detect changes by comparing samples to a reference data set, however, scorecards also require high quantity of RNA for analysis and many of the genes in the panel are no longer relevant. We therefore aimed to develop a method which would allow more flexible, high throughput and cost effective analysis for pluripotent gene expression analysis by applying Fluidigm's dynamic arrays. Dynamic arrays are microfluidic cards in which 48 samples are reacted with 48 assays in a matrix in which each sample is tested against 48 assays, and vice versa.
Here we present the use of dynamic arrays for detection of self-renewal and differentiation into embryonic bodies. Both Dynamic arrays and hSPC scorecards™ results were comparable. However, Dynamic arrays requires 5% of the sample material required for Scorecards™ and the overall cost is reduced by nearly 80%. The advantages of multiplexed matrix analysis with increased robustness for molecular characterisation of cells translates into faster results and cost efficiencies while also allowing customisation for individual cell therapy products.
Nanomedicine Laboratory, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Direct cell labelling is an excellent tool for tracking injected administered cells to determine survival, migration, differentiation and integration when transplanted in host tissues in real time [1]. Optical cell labelling is a non-invasive, sensitive imaging technique that facilitates the development of new cell therapies. Novel optical probes consisting of the clinically approved and used indo-cyanine green (ICG) liposome-based nanoscale system (ICG-Lipo) were synthesised and thoroughly characterised with diameters below 100 nm and high surface positive charge. The liposome construct stabilised the highly liable ICG dye in physiologically-relevant media. Bone marrow derived macrophage cells (BMMs) were extracted from femurs and tibias of male C57Bl/6 mice, with >95% phenotype purity.
BMMs were incubated for 4h with 8 μM of the ICG-Lipo and corresponding controls. The labelling was very efficient with the ICG-Lipo construct compared to the ICG dye alone. Uptake of the ICG-Lipo after 4h was sufficient to obtain an acceptable optical signal for cell tracking, while maintaining cell viability. Intravenous injection of the labelled cells into mice resulted in their accumulation within the lungs in the first few hours with translocation to the liver after 24h. The cell signal was maintained for 7 days in the liver. The accumulation of BMMs in the liver has been suggested for treatment of liver cirrhosis. This study provides a promising method for longer-term optical cell tracking of BMMs in the liver. Further studies will attempt to determine the number of healthy cells that will ultimately reside in the liver.
[1] Calvin S.et al., Stem Cell Rev and Rep, 6 (2010) 317- 333.
Cardiff University, Cardiff, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Adenoviruses (Ad) are the most commonly employed vectors for gene therapy applications. The majority are genetically modified versions of the species C serotype Ad5, which transduce a wide range of cell types via human coxsackie and adenovirus receptor (hCAR), are easy to engineer and propagate to high titres.
The clinical efficacy of Ad5-based virotherapies is suboptimal, partly due to extensive off-target uptake by the liver due to interaction with coagulation factor X (FX), inactivation by pre-existing humoral immunity and lack of tumour-specificity of the vector. We generated a new vector derived from the species D serotype Ad10. The Ad10 genome was captured in a BAC to enable genome modification, the E1 and E3 regions were deleted by recombineering to make it non-replicative, and E4orf6 region was replaced with the Ad5 version to enhance production in 293 cells.
Ad10 was able to transduce cell lines in the presence highly neutralising ascites which completely neutralised transduction by Ad5. Furthermore, Western blots of Ad10 virions showed a substantial reduction of bound anti-Ad antibodies from human serum and ascites compared to Ad5. Ad10 vector did not bind FX, potentially reducing sequestration by the liver in vivo. The cellular receptor for Ad10 is not currently known, however we demonstrate it does not use CD46, but may bind hCAR at lower affinity than Ad5.
Ad10 shows promise as an alternative vector to Ad5. It overcomes a number of the difficulties associated with Ad5 and further capsid modifications may enable targeting to non-native cellular surface receptors.
Bia Separations d.o.o., Ajdovscina, Slovenia
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Adeno-associated virus (AAV) vectors of various serotypes are considered to have high potential for gene therapy applications. Currently, manufacturing of AAV vectors faces the challenge of co-production of incompletely formed particles lacking a recombinant viral genome. Empty capsids increase the dose of total AAV administered for efficient transduction and are thought to cause unwanted immunological reactions against the virus. Removal of empty capsids during manufacturing, as well as analysis of empty/full AAV particle content is therefore a critical requirement for any AAV production process. This poster demonstrates how CIMmultus™ QA monolithic columns can be used to remove empty AAV capsids from the product chromatographically in a single step.
Cobra Biologics Ltd, Keele, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Production of AAV and other viral vectors for cell and gene therapy presents many challenges, a number of which arise as a direct result of difficulties in the production and purification of plasmid DNA. Among those is amplification of unstable secondary structures combined with an exponential increase in demand. Additionally, the ability of AAV to package off-target sequences such as antibiotic resistance genes, presents an important safety consideration for clinical manufacture of AAV vectors.
Cobra Biologics Ltd and Touchlight Genetics Ltd are collaborating on the Innovate UK-funded project aimed at addressing these limitations by evaluating a number of AAV production systems using next-generation DNA technologies. One of such technologies is Touchlight Genetics Ltd's proprietary DNA amplification platform dbDNA™, which eliminates antibiotic resistance and is capable of amplification of unstable secondary structures using an inexpensive benchtop process.
To evaluate dbDNA™ system in AAV2 production, vector yields generated using relevant dbDNA™ constructs were compared to those generated using plasmid based systems. Genomic and total AAV particle titres within the crude cell lysate were determined using qPCR and ELISA assays, respectively. The total to genomic particle ratios were considered as a measure of AAV quality. Similar efficiencies in AAV generation to standard plasmid DNA were observed with these the next-generation DNA constructs, however total to genomic particle ratios when compared to standard plasmid DNA-derived AAV were improved. These findings indicate that novel DNA technologies may be capable of resolving a number of key issues in the manufacture and commercialisation of AAV products.
Department of Genetics and Microbiology, Faculty Of Science, Charles University, Viničná 5, 128 44, Prague 2, Czech Republic
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Polyomaviruses are small icosahedral dsDNA viruses that have been widely investigated as potential gene delivery vectors in the last two decades. Polyomavirus capsid proteins, when produced in heterologous expression systems, self-assemble into virus like particles (VLPs). These VLPs can be loaded with various clinically relevant cargos to serve as vehicles for the intracellular delivery of genes, proteins or chemical compounds. Due to the absence of pre-existing immunity against mouse polyomavirus (MPyV) in the human population, we investigated MPyV as a potential gene and drug delivery vector for anticancer therapy. Similarly, to the wild type virus, MPyV vectors readily enter target cells through the binding of sialic acid, but the efficiency of gene expression by transduced genes is rather low. We identified the restricted capacity of MPyV vectors to escape from the endocytic system as a major bottleneck for their application as gene delivery systems. Here we present data demonstrating that gene transduction efficacy of MPyV vectors can be facilitated by cationic polymers (polyethylenimine) and also, in contrast to the adenovirus vector, by histidine rich peptides. Furthermore, we present the concept of the MPyV vector design that can be applied for cancer cell targeting and cargo release into the cytosol.
The project is suppported by GAČR 305/17-11397S.
Oxford BioMedica (UK) Ltd, Oxford, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Large-scale production of lentiviral vectors (LV) for gene therapy is necessary to meet clinical demand. Oxford BioMedica's platform incorporates a minimal LV system with key safety features such as SIN LTR and codon optimised gag/pol. Historically, manufacture has relied on an adherent, serum-containing process. We have now established a suspension cell, serum-free process up to 200L leading to a significant increase in productivity. This improves the cost of goods and allows the treatment of diseases requiring large quantities of vector. Further enhancements include automation for the generation of LV packaging/producer cell lines; this bespoke Automated Cell Screening System allows the isolation of clones (>1000) by automated limiting dilution cloning utilising robotics. Furthermore, the system can perform routine passage and high-throughput clonal LV production and evaluation. This has generated stable cell lines that produce higher LV yields than transient transfection processes.
For most viral vector platforms, maximal production titres are achieved with ‘inert’ transgenes e.g. GFP. Expression of therapeutic transgenes during production can often adversely affect titres and alter the composition of crude harvest material, potentially requiring bespoke downstream process optimisation. The novel ‘Transgene Repression In vector Production’ (TRiP) system minimises transgene expression during production of both RNA- and DNA-based viral vectors leading to recovery of production titres. This represents a major step towards development of transgene-independent viral vector manufacture.
These advances applied in an industrial scale process bring significant improvements in potency, purity, yield and efficiency which will set the benchmark for commercial LV manufacture.
UCL Great Ormond Street Institute of Child Health, London, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Lentiviral vectors (LV) are being widely applied to deliver therapeutic genes to human cells. High yields of vector stocks are required for most therapeutic applications, driving the need to develop more efficient manufacturing platforms. We hypothesized that generating HIV-1-derived LV may trigger antiviral responses in producer cells and that this might be a limiting factor for large scale campaigns. Cellular antiviral strategies include the induction of innate immune signaling pathways and the activation of mechanisms to epigenetically silence exogenous genetic elements.
We measured the production of inflammatory cytokines and the induction of NF-kB/IFNb/IFIT-1-dependent reporter gene expression as surrogates for innate immune activation in HEK293T cells. Overexpression or depletion of innate signaling molecules and inhibition of viral silencing mechanisms were used to manipulate the intracellular immunity of producer cells. We measured LV titres to assess the effect of innate immune manipulation on LV production.
We show that, in transient systems, the commonly used vector packaging cell line HEK293T is capable of producing a type I IFN response after activation of DNA sensing and inflammatory cytokines were detected in response to transfection of LV expression constructs. However, vector titres were not influenced by either suppression or activation of innate immune pathways or by the presence of exogenous IFN beta. In alternative stable packaging systems, inhibiting epigenetic silencing increased expression of all vector components and improved overall titre. Further understanding of such mechanisms may uncover strategies to improve vector production and lead to more efficient, higher yield vector manufacturing platforms.
Bia Separations d.o.o., Mirce 21, Slovenia
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Plasmid DNA (pDNA) as a pharmaceutical product has stringent requirements of purity and efficacy and often one or more chromatographic steps are used in the downstream process. High ligand density butyl-modified chromatographic monolithic columns are currently used in a polishing step of a pDNA purification process, and are mainly used for separation of supercoiled (sc) pDNA separation from open circular (oc) and linear pDNA isoforms as well as for removal of remaining gDNA and RNA.
This application note presents a comparison of two different polishing processes employing monoliths, namely bind-elute (BE) and the more recently described (2) sample displacement purification (SDP). The results present a 5% increase in pure product yield of sc pDNA alongside with a 60% reduction in chemical consumption and 20% reduction in processing time per gram of product when sample displacement mode is employed.
Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Adenoviruses (Ad) hold promise for a wide range of gene therapy applications. The majority of experimental and clinical studies focus on the hCAR engaging vector, Ad5. To expand the applicability of Ad vectors to a wider range of applications we sought to pseudotype Ad5 vectors with fiber proteins from rare Ad serotypes to exploit the natural diversity of Ad receptor usage.
The primary receptor(s) for species D adenoviruses remain largely unresolved. We used recombineering to generate novel Ad5 vectors pseudotyped with either the fiber (F) or fiber knob (kn) domain from the low seroprevalence species D serotypes Ad10, -15, -24, -29, -48, -53.
Of the generated vectors, only Ad5 and Ad5/kn53 agglutinated red blood cells. Transduction experiments were performed in CHO-CAR and CHO-BC1 cells, expressing hCAR and CD46 respectively. None of the pseudotyped vectors demonstrated increased transduction in CHO-BC1 cells, suggesting CD46 is not utilised. Increased transduction levels in CHO-CAR cells were observed, indicating a potential role for low-affinity hCAR mediated cell entry, although recombinant Ad5 fiber knob protein was only partially able to inhibit this. In the presence of human factor X (FX), transduction of all pseudotypes was significantly enhanced, demonstrating that additional modulation of the hexon:FX pathway may be necessary for targeted intravenous applications.
Due to their alternative tissue tropisms and lower rates of pre-existing immunity, these novel pseudotyped vectors hold promise for various approaches. Further investigation is warranted to evaluate their primary receptor, tropism and the extent of escape from humoral immune recognition.
Nibsc, South Mimms, Potters Bar, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Lentiviral vectors (LVs) are important tools for gene therapy applications as they can integrate into the host cell genome, have relatively large carrying capacity, and more importantly can infect both dividing and non-dividing cells. To improve tropism and vector particle stability in addition to achieving high vector titres, LVs are commonly pseudotyped with vesicular stomatitis virus G-protein Indiana strain (VSVind-G). However, VSVind-G is cytotoxic when expressed continuously. Moreover, it is sensitive to human serum complement, which can limit its in vivo application. Due to these disadvantages, LVs are usually produced transiently. Transient production however, is costly, difficult to scale up and is prone to batch-to-batch variability. Our aim is to develop a stable packaging cell line for LV production using alternative envelope glycoproteins. Cocal virus G-protein (COCV-G) shows around 72% homology on the amino acid level to VSVind-G and has been reported to have a wide cell tropism. In this study, COCV-G-bearing LVs were examined for physical and thermal stability. These vectors are highly stable in both high and low speed centrifugation. Furthermore, they could recover to high titres after several cycles of freeze/thaw and incubation at 37° and 4°C. To investigate which part of G-protein is cytotoxic, chimeric G-proteins based on VSVind-G and COCV-G were generated. Various tests will be performed comparing cytotoxicity amongst different constructs. Moreover, other vesiculovirus envelope glycoproteins will be exploited to find the best available construct for generating stable packaging cell lines based on our continuous LV packaging WinPac system (Sanber, Sci Rep 5:9021 (2015)).
Molecular and Cellular Immunology Section, UCL/GOSH Institute of Child Health, London, UK
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
X-linked inhibitor of apoptosis protein (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency with variable clinical features including haemophagocytic lymphohistiocytosis (HLH) and colitis. Current therapy includes HLH treatment protocols followed by haematopoietic stem cell transplant (HSCT). However, survival in XIAP-deficient patients is significantly lower compared to patients receiving HSCT for other forms of HLH with significant graft versus host disease and disease relapse. Autologous HSC gene therapy could offer an alternative treatment option for these patients. Using a XIAP-knockdown THP-1 cell line we show normalisation of IL-1β levels upon inflammasome activation following lentiviral XIAP gene transfer. Moreover, we are able to demonstrate restoration of TNF-α secretion in response to L18-MDP following XIAP gene transfer in patient monocytes. In a XIAP−/− mouse model, immunization with the Dectin-1 ligand curdlan induces features associated with XIAP-deficiency due to impaired innate immune responses. Serum levels of TNF-α, IL-6, IL-10 and MCP-1 were significantly reduced in XIAP−/− mice 2 hours post-curdlan administration compared to wild-type controls. This profile was reversed after 7 days with XIAP−/− mice secreting higher levels of proinflammatory cytokines alongside splenomegaly. Preliminary data in XIAP-knockdown THP-1 cells challenged with curdlan indicate that TNF-α and IL-1β can be completely restored to control levels following XIAP gene transfer. We now plan to correct immune defects in XIAP−/− mice using lentiviral mediated HSC gene correction to abrogate excessive inflammasome activation following curdlan challenge. In parallel we are developing a targeted gene editing approach for XIAP correction allowing utilisation of endogenous genomic regulatory elements and therefore allow physiological expression of this highly regulated protein.
Gene Transfer Technology group, UCL, London, U.K.
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Preterm birth, associated with greater than 1 million neonatal deaths annually, has no effective treatment. Ascending vaginal infection is believed to be the most common pathway by which bacteria access the uterus to cause preterm delivery. Localised gene delivery of antimicrobial peptides may provide a novel therapy to prevent this infection. To investigate this, we developed two gene technology approaches: 1. Intravaginal administration of bioluminescent bacteria to mimic ascending infection. 2. Cervical overexpression of human β-defensin 3, a potent antimicrobial peptide, using viral gene transfer.
Newcastle University, Newcastle-upon-Tyne, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
The discovery of light-sensitive ion-conducting membrane proteins (channelrhodopsins) in algae was crucial for the foundation of optogenetics, a tool that allows neuroscientists to precisely control neuronal circuitry. Therefore, optogenetic interventions provide attractive means for treating neurological disorders such as epilepsy. The use of optogenetics in clinical applications depends on the delivery of channelrhodopsin-encoding genes to the human brain. Viral vectors represent the most effective means of gene transfer in vivo; lentiviral vectors are commonly used in neuroscience because they infect non-dividing cells (e.g. neurons), stably integrate into the host-cell genome for long-term expression, and do not provoke an inflammatory response.
Our group is developing a lentivirus-based optogenetic gene therapy for the treatment of epilepsy as part of the CANDO project, to be combined with an implantable biomedical device with recording and optical stimulation capability to achieve closed-loop feedback control of seizure activity. We have developed a growing collection of lentivector plasmids, each carrying a different combination of promoter, opsin and reporter gene. Our goal is to provide a range of optogenetic tools for the control of neuronal and glial activity in a targeted manner, by expressing excitatory or inhibitory light-activated ion channels (e.g. cation- or anion-selective channelrhodopsins), in specific cell populations (e.g. using cell-type specific promoters), and with different fluorescent reporters (e.g. EYFP or mCherry). Here we describe the design and manufacture of high-titre third-generation lentiviruses, and a comparison of several methods, including real-time PCR and electron microscopy, for titration and validation of lentiviral vectors before their use in vivo.
UCL, Gene Transfer Technology Group, Institute for Women's Health, London, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Dravet syndrome is an inherited childhood epilepsy, in more than 80% of the cases it can be traced to a mutation in the SCN1A gene, which encodes the voltage-gated sodium channel NaV1.1. It is thought most mutations lead to loss of function and that this disproportionately affects inhibitory neurons. The disease has an incidence of 1:20,000 to 40,000 live births, is characterised by fever-sensitive, refractory and generalized seizures beginning at around six months, followed by psychomotor stagnation and can be associated with early death. There is currently no effective surgical nor pharmacological treatment.
We have previously shown that neonatal intracerebroventricular injections of lentiviral and AAV vectors strongly target the central nervous system and the expression of transgenes persists over development. We therefore generated two vector systems. Firstly, a lentiviral vector carrying the full 6.5kb coding sequence of the SCN1A gene. Previous work has shown that when both halves of a voltage-gated sodium ion channel are expressed separately, they can form a functional protein (Stühmer et al., 1989). Therefore, our second system consists of two AAV vectors each containing half of the gene. Both vectors are driven by a synapsin promoter to achieve neuronal expression.
We have now began testing both approaches in-vitro using immunocytochemistry, ex-vivo electrophysiology, and in-vivo with intracerebroventricular injection to wild type neonatal mice to assess the vector transduction efficacy and possible side effects to ectopic expression. In the future, we will test the vectors in a knock-out mice model, assessing the improvement in seizure frequency, lifespan and brain pathology.
Imperial College London, London, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Cytokines play an important role on tumour growth with a potential anticancer effect through induction of apoptosis and impairing the angiogenic tumour blood vessels; however, their clinical use has been limited due to significant systemic toxicity. Therefore, improvement of cytokine-involved therapy requires efforts to enhance the cytokine delivery, safety, and selectivity to the tumour environment. To achieve a successful therapeutic strategy, we used a hybrid adeno-associated (AAV) phage-based (M13) vector as a safe vehicle for the targeted delivery of therapeutic cytokine genes to cancer cells. This virus is genetically engineered to selectively target the tumour microenvironment as well as the angiogenic blood vessels through the expression of the cyclic RGD4C (CDCRGDCFC) ligand on the bacteriophage capsid to bind to α_v β_3 integrin receptor that is often found to be overexpressed on tumour cells and tumour vasculature. Furthermore, as the M13 bacteriophage lacks the native tropism for mammalian cells, this will add the advantage for safe selective systemic delivery of the vector to the tumour environment. In this study, we showed that the targeted phage-guided delivery of the tumour necrosis factor (TNFα) cytokine induced tumour cell killing and activation of the caspase pathway in medulloblastoma (UW228 and DAOY) and diffuse intrinsic pontine glioma (DIPG), respectively.
1Cancer Phage Therapy group, Centre of Neuroinflammation and Neurodegeneration, Division of Brain Sciences, Department of Medicine/ Imperial College London, Hammersmith Hospital Campus, London, United Kingdom.
Department of Pharmacology, UCL School of Pharmacy, London, UK
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Gaucher Disease (GD) is a lysosomal storage disorder caused by mutations in the GBA gene encoding the enzyme glucocerebrosidase (GCase). Deficiency of GCase causes the accumulation of glucosylceramide in visceral organs and brain. Enzyme replacement therapy is successfully used to ameliorate the visceral pathology, however there is no treatment available for the lethal neurodegeneration. This research focuses on GD type II, the most acute neuronopathic form, in which neuropathology results in death in early infancy. The aim of the project is to intravenously administer an adeno-associated viral vector serotype 9 (AAV9) carrying the functional GBA gene to a GCase-deficient mouse model of GD type II and assess improvement in lifespan, behavior, brain and visceral pathology.
The untreated knock-out mice die 12–14 days after birth from neurodegeneration. AAV9 carrying the functional human GBA gene was intravenously administered into neonatal knock-out mice (n = 5). Treated animals showed increase in their lifespan (p = 0.0081). Since the animals did not develop any evident pathological symptoms, they were sacrificed at 55 days of age for a short-term study. The neuropathology was ameliorated and some of the most affected areas of the brain were partially rescued. Spleen weight, histological analysis, enzymatic assay and blood test revealed improvements in the visceral pathology. Longer-term studies revealed extension in lifespan to at least 180 days of age (approximately 14-fold increase in lifespan).
This minimally invasive pre-clinical proof-of-concept gene therapy study supports potential clinical translation for an acute early-lethal neurodegenerative condition. We are further optimising our vector for maximum therapeutic efficacy.
Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Institute of Cardiovascular & Medical Sciences, The University of Glasgow, Glasgow, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
BioReliance, Glasgow, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
As modern technologies and scientific advancements change the landscape of medicinal biological product development and manufacture, along with it we must consider how we look at safety. From characterisation of novel cell substrates and gene therapy vectors to screening of raw materials and final products, Next Generation Sequencing (NGS) methods have demonstrated unbiased and sensitive detection of contaminating viral agents and are likely to play an increasing role in evaluating safety of advanced biological therapies in the future.
Detailed characterization of viral vectors used in the manufacture of vaccines and genetic therapy products is required. This characterization includes the genome identification/confirmation of a viral seed stock (VSS) and the investigation of sequence variants (subpopulation) in that stock. Current guidelines from the FDA and the WHO recommend full-length sequencing of viral vectors in order to assure the absence of sequence variants or subpopulations.
Next Generation Sequencing (NGS) can be used to confirm the identity of an exogenous sequence (insert) in a viral vector and can perform deep, whole viral genome sequencing making it the most effective means to assure the absence of sequence variants or subpopulations in a viral vector. When combined with a precise algorithm, NGS generated data from the VSS sample tested for identity can also be used to detect and identify the sequencing signature of any adventitious agents present.
This presentation considers risk mitigation approaches to control potential viral contamination in biological products and looks at case studies in which Next Generation Sequencing is being used in this area.
Department of Pathology, University of Cambridge, Cambridge, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Type 1 Diabetes (T1D) is an autoimmune disease characterised by the pancreatic infiltration of immune cells resulting in T cell-mediated destruction of the insulin-producing beta cells. Steady glycaemic control is crucial for T1D patients, however it remains difficult to achieve with exogenous insulin therapy. When left untreated, prolonged hyperglycaemia can lead to micro and macrovascular complications and neuropathy. Conversely, insulin overtreatment may cause hypoglycaemia, which can lead to neurological disorders or death. Therapeutic advantages conferred by approaches alternative to insulin administration are being investigated but long-term safety and efficacy data are lacking. An interesting and unexplored option is the use of genetically modified adeno-associated virus (AAV) vectors that have proved successful in the cure of monogenic disorders and have shown negligible toxicity and immunogenicity. By taking advantage of a large collaboration involving groups in Singapore, London and Cambridge we have investigated ways of restoring a basal level of insulin production in various diabetic animal models via administration of liver tropic AAV8 vector containing a codon-optimised human insulin gene. Our results indicate that no significant pre-existing immunity against AAV8 vector was found in the tested models before gene therapy and the transgene was successfully expressed. However, the titer of anti-AAV8 antibodies increased over time after vector administration, indicating mounting humoral immune response vs AAV8 but not against the insulin transgene product. However, diabetes management proved challenging in the presence of an ongoing spontaneous autoimmune disease, suggesting that a pre-existing immune condition could impair the success of the therapy.
Department of Pharmacology, University College London, London, United Kingdom
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
Infantile neuroaxonal dystrophy (INAD) is a debilitating, intractable and ultimately lethal neurodegenerative disorder. It is caused by mutations in the PLA2G6 gene that encodes for phospholipase A2. Patients present neurodegeneration-associated symptoms between six months and three years of age. Severe spasticity, progressive cognitive decline, and visual impairment typically result in death during the first decade (Morgan et al, 2006). There is no disease-modifying treatment available and palliative care focuses on quality of life. Therefore, there is an overwhelming need to develop novel therapies to treat INAD patients.
Additionally, we aim to develop an AAV-mediated gene therapy approach for the treatment of INAD and conduct a pre-clinical study in the pla2g6-inad mouse model. The objective is to be able to prevent or ameliorate both the peripheral nervous system and the central nervous system phenotype and improve the lifespan and/or quality of life of the mouse. We will use recombinant adeno-associated virus serotype 9 vector (AAV9) to deliver therapeutic human PLA2G6 gene to the neonatal pla2g6-inad mouse CNS via either intracranial or intravenous administration. The synapsin-I promoter, a strong neuronal promoter, will drive the human PLA2G6 gene.
A preliminary study was carried out on the pla2g6-inad mouse model, where neonatal pla2g6-inad mice were administered 8.9x1013 vector genomes of AAV9.hSYN1.hPLA2G6 via intracranial injections. Treated pla2g6-inad mice showed an increased lifespan (n = 5; median survival of 173 days; P < 0.001) compared to untreated pla2g6-inad mice (n = 5; median survival of 98 days). The significant increase in lifespan supplemented with significant improvements in behavioural tests validates the potential beneficial use of gene therapy for infantile neuroaxonal dystrophy (INAD).
BSGCT Exhibition, drinks & poster judging, Exhibition, poster and catering areas, April 20, 2017, 6:00 PM – 7:15 PM
The UK Stem Cell Bank (UKSCB) is a key component of the UK regenerative medicine infrastructure charged with procuring, processing (banking and testing) and distributing seed stocks of human embryonic stem cell (hESC) lines for research and human application. One of the key objectives of the UKSCB is to bank and release a panel of carefully selected stem cell lines that meet the EU Tissue and Cell Directives (EUTCD) as set out in Human Tissue Authority (HTA) regulations. These directives set a benchmark for the standards that must be met when carrying out any activity involving tissues and cells for human application. All cells are subject to ethical scrutiny by a national Steering Committee, following which the UKSCB performs a due diligence protocol which establishes for each individual cell line, whether it could meet the requirements of the EUTCD and is thereby provide a starting material for clinical trials.
Currently, 38 hESC lines have been approved for deposit as EUTCD-Grade by the UK Steering Committee and at present, 3 lines have been banked and released. Three more lines are currently undergoing banking and rigorous testing to ensure future release of seed stocks that are well-characterised and of high quality. Here we describe the key elements of the UKSCB process for these lines including review, processing, characterisation, storage and distribution; all of which are designed to meet the requirements of the UKSCB's regulatory licence which has been maintained successfully for more than 10 years.
This current programme of work represents the start of a pipeline of EUTCD-grade hESC lines to support the regenerative medicine community in the development of quality and safety-assured cell therapies. These cell lines are now available for research or commercial development internationally, under a non-exclusive license.