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Novel transfection agents as tools for biology

Primary Supervisor: Dr Paco Fernandez-Trillo (PFT) Secondary supervisor: Robin C May (RCM) and Sara Jabbari (SJ)

PhD project title: Novel transfection agents as tools for biology

University of Registration: University of Birmingham

Project outline:

Genetically engineered cells are valuable tools in cell biology and biotechnology. 1 The ability to express foreign genes (such as GFP) within cells is critical to modern biological research. However, current methods to engineer cell lines, such as retroviral infection, electroporation, or direct microinjection of the vector into the nucleus, have several disadvantages, including the cost associated or the stress induced in the cell. As such, new materials and technologies are required that can efficiently transport genetic information across cellular barriers without damaging these cell lines. We have recently developed an innovative screening technology, that allow us to identify transfection agents in a timely and efficient way. 2 This methodology allow us to screen for transfection efficiency and toxicity without having to purify any of the candidate transfection agents, simplifying this way the discovery process.

In this multidisciplinary project, we will identify novel transfection agents capable of delivering plasmid DNA to cells that are classically thought of as difficult or impossible to transfect (such as macrophages or neuronal cells).

Research Objectives: Our high-throughput strategy is based on the modification of a polymer scaffold (P1) to give us access to a library of modulated transfection agents (PM) with a broad range of chemical functionalities in highly efficient way (Scheme). Chemical functionality will be selected from a range of moieties known to mediate conjugation to nucleic acids (i.e. cationic residues) and transport acrosse membranes (i.e. hydrophobic moities). Detailed objectives are:

O1. Synthesis of Polymer Scaffolds (P1): In this part of the project we will explore the use of other polymer backbones (e.g. degradable polyesters, helical polyacetylenes) and funcionalities (e.g. alkoxyamine) that can improve biocompatibility and stability of the formed hydrazones. All new compounds will be characterised by NMR, HRMS, GPC and IR.

O2. Synthesis of Modulated Transfection agents (PM): We will prepare candidate polymers Pm simply by incubation of P1 with the desired aldehydes, and the activity tested by diluting this mixture in the relevant assay. Loading efficiencies will be evaluated by NMR and GPC, and the abilily of PM to facilitate the transport of nucleic acids across membranes monitored first with a fluorogenic assay, to identify any candidate polymers with potential to damage membranes.

O3. High-throughput evaluation of Transfection: Here we will use J774 macrophages representative cell line for which improvement in the levels of transfection are needed. First, toxicity of P1 and PM to this cell line will be evaluated using a range of assays (e.g. trypan blue, LDH). Then, the ability of PM to deliver pGFP to this cell line will be evaluated. We will employ an iterative process so that toxicity and transfection efficiency will inform the choice of polymer scaffolds (P1), aldehydes and their compositions. Mathematical modelling will be employed to correlate how these parameters affect transfection and toxicity and optimise PM design. Overall, we aim to identify a new transfection agent that improves on current levels of transfection for this cell line (∼30%) without triggering toxicity or stress.

BBSRC Strategic Research Priority: Renewable Resources and Clean Growth: Industrial Biotechnology

Techniques that will be undertaken during the project:

Due to the multidisciplinary approach, the PhD student will develop a wide range of experimental skill described in the following training objectives:

Training 1. Cell culture and cell phenotyping including culture of J774 macrophages and primary myeloid cells.

Training 2. Fluorescence and Confocal Microscopy including live cell imaging, multidimensional imaging and quantitative fluorescence work

Training 3. Computational Modelling: The skills developed as part of the Foundation Skills Modules (i.e.: Large Dataset Handling, Quantitative Biology) will be supported by training in multinomial logistic and multivariate regression.

Training 4. (Polymer) synthesis and Characterisation: Controlled polymerisation (i.e. free radical), combinatorial chemistry, (bio)conjugation using efficient chemistries (i.e. hydrazine-aldehyde condensation), polymer characterisation (NMR, GPC, IR). This expertise will be key for the completion of Objective 1.

Contact: Dr Paco Fernandez-Trillo, University of Birmingham