In vitro transcribed mRNA therapies for Primary Ciliary Dyskinesia

Background:

Messenger RNA therapies offer great potential for a range of disease applications form vaccines such as COVID-19, to rare diseases. The great advantages of mRNA as a therapeutic compared to plasmid DNA or viral vectors, are its high level of activity, ease of production, and safety as there is no risk of genomic integration. Primary ciliary dyskinesia (PCD) is a genetic disease caused by mutations in genes affecting the structure and movement of the cilia lining the upper airways.

Aims/Objectives:

We aim to develop mRNA therapies for PCD focusing on the development of the mRNA encoding CCDC39 and CCDC40; models and assays for testing the therapy and methods of delivery.

1. Evaluate mRNA design for optimal expression level and duration in the airway epithelium with respect to the 5’-cap, tissue-optimised 5’-untranslated region (5’-UTR), 3’-UTR and polyA.

2. Optimise coding sequence for CCDDC39 and CCDC40 and test expression.

3 Optimisation of nanoparticle design for delivery of mRNA including packaging for extracellular stability and intracellular deposition in the cytoplasm within the cell.

4. Evaluation of transfection efficiency in specific cell types in air-liquid interface cultures of epithelial cells with CCDC39 or -40 mutations.

Methods:

The student will learn methods related to;

1. In vitro transcription production of mRNA including design, sub-cloning and production of the plasmid template by bacterial protocols.

2. Protocols for production of mRNA by In vitro transcription including purification and analytics.

3, Air liquid interface (ALI) culture methods, transfection and methods for detection of PCD mRNA uptake in to cells and protein expression (e.g Western, ELISA).

4. Preparation of nanoparticles for transfection of ALI cultures.

5. Functional assays for correction of ciliary structure and motility, such as high speed video microscopy.

Timeline (if applicable):

Year 1 Aim s 1-2,; Year 2 Aims 2-3, Year 3: Aims 3-4, and write up.

References:

1. … Legendre, M., L.E. Zaragosi, and H.M. Mitchison, Motile cilia and airway disease. Semin Cell Dev Biol, 2021. 110: p. 19-33
2. Horani, A. and T.W. Ferkol, Advances in the Genetics of Primary Ciliary Dyskinesia: Clinical Implications. Chest, 2018. 154(3): p. 645-652.
3. … Abdelhamed, Z., et al., A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice. Development, 2018. 145(1).
4. … Mauger, D.M., et al., mRNA structure regulates protein expression through changes in functional half-life. Proc Natl Acad Sci U S A, 2019. 116(48): p. 24075-24083.
5. … Zylberberg, C., et al., Engineering liposomal nanoparticles for targeted gene therapy. Gene Ther, 2017. 24(8): p. 441-452