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Unravelling cell entry and intracellular trafficking of adenoviruses at the nanoscale using coherent optical nanoscopy


   Cardiff School of Biosciences

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  Prof P Borri, Prof W Langbein  No more applications being accepted  Competition Funded PhD Project (Students Worldwide)

About the Project

Adenoviruses (AdV) are attracting strong interest as vectors for gene therapies (including the COVID-19 vaccine) and as oncolytics for cancer treatments. To fulfil its role as a vector, an AdV needs to successfully deliver its DNA genome to the host nucleus. Notably, AdV infection is a complex process involving several steps: binding to a primary receptor at the cell surface, internalization via endocytosis, escape from the endosome, intracellular trafficking and finally genome delivery to the nucleus. AdV exist in many serotypes, some of which have been well characterised (for example Ad5) while others are unstudied and might offer exciting new avenues for “virotherapies”. Importantly, it has emerged that different serotypes undergo different trafficking pathways, but the reasons are often unclear [1]. Yet, when considering the use of AdV as vectors for therapeutic applications, understanding how they traffic inside cells and their exit efficiency from endosomes is key to the design of constructs for optimal transgene expression in target cells. Moreover, although AdV are considered not to be very dangerous, adenoviral infection is a significant cause of mortality in the immunocompromised individual, so understanding their means of infection is also critical for developing new antivirals to treat such patients.

A powerful way to unravel the journey of viruses is to directly follow individual virions in space and time inside living cells, using light microscopy and single particle tracking (SPT) techniques [2]. The majority of SPT methods to date exploit fluorescent probes labelling the virion of interest. However, all organic probes are prone to photobleaching which severely limits the total time window of tracking (to at best few minutes) and the precision with which the probe can be localised in space. Moreover, photobleaching is often accompanied by cytotoxicity. The lead supervisor lab has pioneered a new SPT technology using photostable gold nanoparticle (AuNP) probes, which overcomes all limitations of fluorescent-based SPT. The technique exploits the strong and colour-selective absorption and scattering of light of a AuNP. Using a combination of short optical laser pulses to generate and detect changes in the AuNP transmission/scattering (via a process called four-wave mixing – FWM), the technique is uniquely sensitive to single small AuNPs which are detected background-free in 3D with localisation precision at the nanoscale inside cells [3]. Notably, AuNP are well established probes in electron microscopy. Hence the technique lends itself for correlative light electron microscopy (CLEM) using the same AuNP, to pinpoint the probe location within the cellular ultrastructure with (sub)-nm spatial resolution.

This project has two main interconnected aims: 1) to develop AdV tagged with small gold nanoparticles and 2) to unravel the cell entry and intracellular trafficking of AdV by SPT with unprecedented spatio-temporal resolution using FWM-CLEM. The co-supervision (Parker) has expertise with various AdV serotypes (including Ad5, Ad10 and Ad49) and has developed biochemical tools to selective label the AdV fibre knob proteins with AuNPs. The co-supervisor in Bristol (Verkade) is a leader in CLEM. Together with the Lead supervisor (Borri) and the co-supervisor (Langbein), they have recently demonstrated a proof-of-principle FWM-CLEM workflow which will be exploited in this project. Areas in which the student will be able to take ownership and steer the project include engineering AdV serotypes with AuNP also inside the capsid (e.g. at the terminal protein), and designing experiments to gain a mechanistic understanding of the intracellular trafficking and genome delivery of new AdV serotypes.

Additional Supervisors - Prof Alan Parker ([Email Address Removed]) and Prof Paul Verkade ([Email Address Removed]

About the GW4 BioMed2 Doctoral Training Partnership

The partnership brings together the Universities of Bath, Bristol, Cardiff (lead) and Exeter to develop the next generation of biomedical researchers. Students will have access to the combined research strengths, training expertise and resources of the four research-intensive universities, with opportunities to participate in interdisciplinary and 'team science'. The DTP already has over 90 studentships over 6 cohorts in its first phase., along with 20 students in its second phase. 

Eligibility

Residency: The GW4 BioMed2 MRC DTP studentships are available to UK and International applicants. Following Brexit, the UKRI now classifies EU students as international unless they have rights under the EU Settlement Scheme. The GW4 partners have all agreed to cover the difference in costs between home and international tuition fees. This means that international candidates will not be expected to cover this cost and will be fully funded but need to be aware that they will be required to cover the cost of their student visa, healthcare surcharge and other costs of moving to the UK to do a PhD. All studentships will be competitively awarded and there is a limit to the number of International students that we can accept into our programme (up to 30% cap across our partners per annum).

Academic criteria: Applicants for a studentship must have obtained, or be about to obtain, a UK degree, or the equivalent qualification gained outside the UK, in an appropriate area of medical sciences, computing, mathematics or the physical sciences. Please check the entry requirements of the home institution for each project of interest before completing an application. Academic qualifications are considered alongside significant relevant non-academic experience.

English requirements: If English is not your first language you will need to meet the English language requirements of the university that will host your PhD by the start of the programme. Please refer to the relevant university for further information.

How to Apply

A list of all the projects and how to apply is available on our website at gw4biomed.ac.uk. You may apply for up to 2 projects.

Please complete an application to the GW4 BioMed2 MRC DTP for an ‘offer of funding’. You may also need to make an application for an 'offer to study' to your chosen institution(s) – further details are on the website.

Please complete the online application form by 5.00pm on Wednesday, 2nd November 2022. If you are shortlisted for interview, you will be notified by Friday 16th December 2022. Interviews will be held virtually on 25th and 26th January 2023.


Funding Notes

The GW4 BioMed2 MRC DTP is offering up to 20 funded studentships across a range of biomedical disciplines, starting October 2023
The four-year studentship provides funding for fees and stipend, as well as other research training and support costs, and are available to UK, EU, and International students.
Funding consists of UK tuition fees, as well as a Doctoral Stipend matching UK Research Council National Minimum (£16,062 p.a. for 2022/23, updated each year). Additional research training and support funding of up to £5,000 per annum is also available.
Part time study is also available.

References

1]10.3390/pharmaceutics13101585 [2] 10.1021/acs.chemrev.9b00692 [3]10.1039/C9NR08512B
Further Information
For informal enquiries, please contact GW4BioMed@cardiff.ac.uk
For project related queries, please contact the respective supervisors listed on the projects.

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