Developing novel approaches to gene therapy cystic fibrosis

Background:
The genetics of cystic fibrosis is very well characterised with six classes of CFTR mutations. Personalised medicines called CFTR modulators have been developed for some CFTR mutations. but  approximately 10% of patients have mutations that are not amenable to CFTR modulators and currently remain without the prospect of any treatment. These patients have mutations where no protein is produced.

In vitro transcribed mRNA (IVT mRNA) is a promising approach for CFTR protein replacement. Advantages of mRNA include the cytoplasmic location of the translational machinery, pre-empting the need for nuclear uptake, which is one of the main barriers limiting pDNA efficiency, and so greatly enhances transfection. Messenger RNA therapeutics are also safer as, unlike pDNA, they lack the potential for genomic integration and insertional oncogenesis.

Aims/Objectives:
The aim of this project is to develop a therapy for CF lung disease based on CFTR messenger RNA delivered by an aerosolised nanoparticle formulation designed to overcome the mucociliary barriers to transfection of the airways in the following studies:

1. Develop targeted nanoparticle formulations for mRNA delivery
2. CFTR mRNA transfection of ALI cultured CF cells
3. Murine airway transfection with mRNA

Methods:

1. Develop nanoparticle formulations for mRNA delivery (Year 1)
The student will develop improved nanoformulations for airway delivery that are better equipped to overcome the barriers to lung delivery, such as mucus and achieve targeted uptake into the epithelium. Experiments will be performed to characterise the nanoparticles size, charge and packaging efficiency and, their mucus penetration and transfection efficiency in cell culture.

2. CFTR mRNA transfection of ALI cultured CF cells (Year 2)
Transfection of lung epithelial cells with fluorescently labelled mRNA formulations will be optimised and analysed by fluorescence microscopy and flow cytometry while quantitative RT-PCR will be performed to quantify mRNA uptake. CFTR protein production will be confirmed by Western blotting. Restoration of CFTR expression and functional  effects on the CF ALI culture physiology will be performed by electrophysiology experiments, correction of mucociliary properties.

3.  Murine transfection studies ( Year 3)
We propose to compare optimal formulations from above for efficiency and inflammatory responses in mice transfected with luciferase and GFP reporter mRNA and compared with their corresponding pDNAs for overall expression (luciferase) and cell percentage and location (GFP). Histological and molecular analysis of CFTR mRNA protein expression will be performed. Lung sections will also be stained with haematoxylin and eosin for analysis of inflammation and bronchiolveolar lavage fluids will be analysed for inflammatory cells.

References:
1.  F. Ratjen et al., Cystic fibrosis, Nat Rev Dis Primers 1 (2015) 15010;
2.  U. Griesenbach et al., Cystic Fibrosis Gene Therapy in the UK and Elsewhere, Hum Gene Ther 26(5) (2015) 266-75;
3.  U. Sahin et al., mRNA-based therapeutics--developing a new class of drugs, Nature reviews. Drug discovery 13(10) (2014) 759-80; 
4.  W.F. Lai, W.T. Wong, Design of Polymeric Gene Carriers for Effective Intracellular Delivery, Trends Biotechnol 36(7) (2018) 713-728.
5.  A.D. Tagalakis, et al., Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung, Thorax 73(9) (2018) 847-856