D.Phil Studentships in the Radcliffe Department of Medicine

We currently the following D.Phil projects available via the Radcliffe Dep of Medicine's Scholars Programme (read this first).

1. Clinical Lentiviral vector design and production

Dr Stephen C. Hyde   

Project: Lentivirus is emerging as an exciting gene therapy vector for clinical applications. Lentiviral vectors show an increased safety profile compared with standard gamma retroviruses currently used in the clinic, and early studies in animal models indicate that these vectors can be repeatedly administered without loss of efficacy.

Some clinical successes with lentiviral vectors have been reported in small numbers of patients following delivery to the eye (to treat blindness) and brain (Parkinson's disease). Whereas the quantities of virus needed to treat these organs is relatively small, applications to treat lager organs will require much larger quantities of virus produced to a clinical standard. Until we are able to produce these vectors at high yield, this bottleneck will prevent clinical application for diseases such as haemophilia, lysosomal storage disorders, and cystic fibrosis.

We are therefore developing a scaleable production technology for human and simian lentiviral vectors using cell growth in suspension. Much is already known about lentivirus vector design and function due to work with HIV, but this has not yet been applied to improving virus production. In this project we would seek to understand and manipulate the key factors involved in increasing viral cell burst to improve viral yield, which may incorporate aspects of: the design of the viral vector genome; the codon-optimisation of viral and therapeutic sequences; enhanced transfection conditions for expression of viral components; and the ratio of expressed virus coat proteins for pseudotyping.

In addition, changes to the viral vectors need to be compliant with European Pharmacopoeia guidance regarding the use of these vectors in humans. Novel changes in vector design and production methodology is expected to generate intellectual property to support new clinical applications.

2. CG dinucleotide involvement in repeated airway administration of Lentiviral vectors to the lung^Top

Dr Deborah R. Gill   

The selection of a gene therapy vector for a particular disease application is crucial to success, with the first step being the choice between viral and non-viral vectors. The use of viral vectors exploits the natural efficiency of viruses to infect host cells, which has evolved over time, but their use is limited by the generation of host immune responses. Non-viral, also known as synthetic, vectors appear less efficient, but generate only minimal immune responses resulting in the capacity for repeated application without loss of efficacy. The latter advantage is particularly important for treatment of chronic diseases where the target cells are terminally differentiated and are slowly replaced.

It has recently been reported that some types of lentiviral vector can be repeatedly administered to the airways without loss of efficacy. This is the first viral vector for which repeated administration has been demonstrated and the reasons are not well understood. This project will investigate factors that facilitate successful repeat administration of lentiviral vectors pseudotyped with airway-specific coat proteins, compared with those vectors that cannot be repeatedly administered. Key factors may include the origin of the Lentivirus vector, particularly SIV versus HIV-derived constructs, the dose of virus delivered and the host species and route of application. Importantly, the cell types targeted for expression and the degree of expression in 'off-target' cells could be crucial. Lentiviral transduction of plasmacytoid dendritic cells can result in production of Type I Interferons via triggering of endosomal Toll-like receptors 7 and 9, although the specific activating ligand is not proven.

In particular we are interested in the role of CG dinucleotides present in plasmid DNA precursor molecules used in the production of lentivirus particles. These plasmids, expressing various essential and non-essential viral components appear, under certain circumstances, to be carried over into the final viral capsid, and are thus delivered to target organs where they may affect the host response to the virus.

Understanding the key aspects of lentiviral vector interaction with host cells should facilitate the use of these vectors in treating chronic lung diseases such as cystic fibrosis, emphysema and COPD, which will ultimately require repeated administration to the patient. Furthermore these studies may inform strategies using gene transfer vectors to develop effective vaccination for infectious diseases and cancer immunotherapy.


3. Airway specific pseudotyping of lentiviral vectors – engineering the ultimate lung gene therapy vector^Top

Dr Stephen C. Hyde   

Early clinical trial data suggest that lentiviral vectors can provide safe and efficacious treatments for diseases of the brain, eye and hematopoietic system. Regrettably, similar vectors have poor tropism for airway cells, which has hindered their development for the treatment of lung diseases. Interestingly, gene transfer vectors derived from murine paramyxovirus (Sendai virus) show remarkably efficient airway gene transfer – modest viral doses can transduce essentially 100% of the murine conducting airway.

This astonishing efficiency is in part due to the Sendai virus F and HN coat proteins that facilitate rapid and efficient airway cell entry. Unfortunately, while Sendai virus vectors promote highly efficient lung gene transfer, they are also inflammatory and immunogenic – properties that have slowed their development for therapeutic gene transfer. However, a powerful property of lentiviral vectors is the ability to modify the viral tropism by changing the viral coat protein – a process termed viral pseudotyping. Replacing the widely used VSV-G pseudotyping protein with the F and HN proteins from Sendai virus generates a lentiviral pseudotype with improved tropism for airway cells. We are developing F/HN pseudotyped lentiviral vectors for the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD), emphysema and cystic fibrosis (CF). One disadvantage of this novel pseudotype is that the F and HN proteins are less efficiently incorporated into the viral capsid compared with conventional pseudotyping proteins, which hinders both the efficacy of the virus and the manufacturing of F/HN pseudotyped lentiviral vectors.

The aim of this project is to develop novel hybrid F and HN proteins where their cytoplasmic tails (the region that interacts with the lentiviral gag protein core) are deleted, modified or replaced with the cytoplasmic tails of more efficient pseudotyping proteins such as VSV-G and HIV gp41. This project would combine our knowledge of airway cell uptake, transmembrane structure, GMP compliant vector development, transgene expression cassette design including codon-optimisation and lentiviral manufacturing. Optimisation of the F and HN pseudotyped lentiviral vector will facilitate more efficient airway gene transfer and vector production, and provide a versatile platform technology for lung gene delivery.


4. Development of plasmid DNA-based mucosal vaccines^Top

Dr Deborah R. Gill   

The adult human mucosal surface, comprising the surfaces of the respiratory, digestive and genitourinary tracts, covers an immense area (~400m squared) that is vulnerable to infection by pathogenic microorganisms. Intense immune surveillance, provided by an integrated network of lymphoid and non-lymphoid cells and effector molecules, occurs at these surfaces. Mucosal immune responses are most efficiently induced by the administration of vaccines onto mucosal surfaces, but most licensed vaccines are administered by injection and fail to elicit protective mucosal immunity.

This focus on parenteral vaccine administration is understandable: a known quantity of antigen is easily delivered and the generation of antigen specific antibodies and lymphoid cells are readily measured in blood samples. In contrast, the development of mucosal vaccines has lagged behind, in part because the administration of mucosal vaccines and the measurement of mucosal immune responses are more complex. For example, the efficiency of vaccine delivery and the levels of antibodies in low volume mucosal secretions are difficult to quantify, and the recovery of mucosal T-cells is technically challenging.

Genetic vaccines based on plasmid DNA (pDNA) have several advantages over conventional vaccine designs: they eliminate the infection risks associated with attenuated viral vaccines, present antigens to both MHC class I and class II molecules, have an excellent storage and shipping profile and can be rapidly developed to combat an emerging pathogen. Consequently, recent concerns regarding potential pandemic respiratory diseases and respiratory biological weapons have led to increased interest in pDNA vaccines. Disappointingly, pDNA vaccine development for such targets appears to be limited by the potency of available gene transfer formulations.

However, we have recently developed a potent airway gene transfer formulation that possesses two key desirable properties for a mucosal pDNA vaccine: it directs efficient expression of airway antigens following topical administration to the nasal epithelium and/or aerosol administration to the lungs, and it is readily manufacturable to GMP standards. This project will involve the design of pDNA expression vectors that both maximally express experimental antigens and potently engage with the Toll-like receptor pathways to facilitate stimulation of mucosal immune responses. In addition, the project will include the further development of gene transfer formulations to enhance potential vaccine stability, and lung aerosol delivery studies to optimise vaccine dose.

What training will be provided?^Top

This project is based in the Gene Medicine Research group within the John Radcliffe Hospital, which focuses mainly on translational gene therapy for lung diseases.

Students will be exposed to all aspects of gene therapy research from basic science through therapeutic evaluation in model systems and into to clinical trials. The successful student would receive specific training in: molecular biology, cell culture, PCR, FACS, Western blotting, immunocytochemistry, ELISA, Quantitative (RT)-PCR, lentivirus production, & Tangential Flow Filtration (TFF) methods.

The student can attend the Methods & Techniques training course based in the nearby Weatherall Institute of Molecular Medicine, as well as internal and guest speaker programmes.

All students are encouraged to enrol in training workshops within the Medical Sciences Division focusing on generic and transferable skills for career progression, including scientific writing and presentation skills, ethics, intellectual property, statistics, etc.

How to apply to the RDM Scholars Programme.^Top

Applications for this D.Phil Programme will be handled centrally by the department rather than by our group.

Applicants can select up to three different project choices from those available (each of these projects above count individually).

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